Treating bacteria with electric fields

ABSTRACT

Cells that are in the process division are vulnerable to damage by AC electric fields that have specific frequency and field strength characteristics. The selective destruction of rapidly dividing cells can therefore be accomplished by imposing an AC electric field in a target region for extended periods of time at particular frequencies with particular filed strengths. Some of the cells that divide while the field is applied will be damaged, but the cells that do not divide will not be harmed. This selectively damages rapidly dividing cells like bacteria, but does not harm normal cells that are not dividing. Since the vulnerability of the dividing cells is strongly related to the alignment between the long axis of the dividing cells and the lines of force of the electric field, improved results can be obtained when the field is sequentially imposed in different directions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.12/958,623, filed Dec. 2, 2010. Application Ser. No. 12/958,623 claimsthe benefit of U.S. provisional application 61/266,392, filed Dec. 3,2009; and application Ser. No. 12/958,623 is also a continuation-in-partof U.S. patent application Ser. No. 12/146,918, filed Jun. 26, 2008,which (1) claims the benefit of U.S. provisional application 60/947,212,filed Jun. 29, 2007, and (2) is also a continuation-in-part of U.S.patent application Ser. No. 11/111,439, filed Apr. 21, 2005, which (a)claims the benefit of U.S. provisional application 60/565,065, filedApr. 23, 2004, and U.S. provisional application 60/639,873, filed Dec.27, 2004; (b) is a continuation-in-part of U.S. patent application Ser.No. 11/074,318, filed Mar. 7, 2005, which is a continuation-in-part ofU.S. patent application Ser. No. 10/315,576, filed Dec. 10, 2002, whichis a continuation-in-part of U.S. patent application Ser. No.10/285,313, filed Oct. 31, 2002, which is a continuation-in-partapplication of U.S. patent application Ser. No. 10/263,329, filed Oct.2, 2002; (c) is a continuation-in-part of U.S. patent application Ser.No. 10/402,327, filed Mar. 28, 2003, which is a continuation-in-part ofU.S. patent application Ser. No. 10/204,334, filed Oct. 16, 2002, whichis the U.S. national phase of PCT/IB01/00202, filed Feb. 16, 2001, whichclaims the benefit of U.S. provisional application 60/183,295, filedFeb. 17, 2000; and (d) is a continuation-in-part of U.S. patentapplication Ser. No. 10/288,562, filed Nov. 5, 2002, which claims thebenefit of U.S. provisional application 60/338,632, filed Nov. 6, 2001.Each of the above-referenced applications is hereby incorporated byreference in its entirety.

TECHNICAL FIELD

This invention concerns selective destruction of rapidly dividing cellsin a localized area, and more particularly, selectively destroyingtarget cells without destroying nearby non-target cells by applying anelectric field with specific characteristics in vitro or to a region ina living patient.

BACKGROUND

All living organisms proliferate by cell division, including cellcultures, microorganisms (such as bacteria, mycoplasma, yeast, protozoa,and other single-celled organisms), fungi, algae, plant cells, etc.Dividing cells of organisms can be destroyed, or their proliferationcontrolled, by methods that are based on the sensitivity of the dividingcells of these organisms to certain agents. For example, certainantibiotics stop the multiplication process of bacteria.

The process of eukaryotic cell division is called “mitosis”, whichinvolves nice distinct phases (see Darnell et al., Molecular CellBiology, New York: Scientific American Books, 1986, p. 149). Duringinterphase, the cell replicates chromosomal DNA, which begins condensingin early prophase. At this point, centrioles (each cell contains 2)begin moving towards opposite poles of the cell. In middle prophase,each chromosome is composed of duplicate chromatids. Microtubularspindles radiate from regions adjacent to the centrioles, which arecloser to their poles. By late prophase, the centrioles have reached thepoles, and some spindle fibers extend to the center of the cell, whileothers extend from the poles to the chromatids. The cells then move intometaphase, when the chromosomes move toward the equator of the cell andalign in the equatorial plane. Next is early anaphase, during which timedaughter chromatids separate from each other at the equator by movingalong the spindle fibers toward a centromere at opposite poles. The cellbegins to elongate along the axis of the pole; the pole-to-pole spindlesalso elongate. Late anaphase occurs when the daughter chromosomes (asthey are now called) each reach their respective opposite poles. At thispoint, cytokinesis begins as the cleavage furrow begins to form at theequator of the cell. In other words, late anaphase is the point at whichpinching the cell membrane begins. During telophase, cytokinesis isnearly complete and spindles disappear. Only a relatively narrowmembrane connection joins the two cytoplasms. Finally, the membranesseparate fully, cytokinesis is complete and the cell returns tointerphase.

In meiosis, the cell undergoes a second division, involving separationof sister chromosomes to opposite poles of the cell along spindlefibers, followed by formation of a cleavage furrow and cell division.However, this division is not preceded by chromosome replication,yielding a haploid germ cell. Bacteria also divide by chromosomereplication, followed by cell separation. However, since the daughterchromosomes separate by attachment to membrane components; there is novisible apparatus that contributes to cell division as in eukaryoticcells.

It is well known that tumors, particularly malignant or canceroustumors, grow uncontrollably compared to normal tissue. Such expeditedgrowth enables tumors to occupy an ever-increasing space and to damageor destroy tissue adjacent thereto. Furthermore, certain cancers arecharacterized by an ability to transmit cancerous “seeds”, includingsingle cells or small cell clusters (metastases), to new locations wherethe metastatic cancer cells grow into additional tumors.

The rapid growth of tumors, in general, and malignant tumors inparticular, as described above, is the result of relatively frequentcell division or multiplication of these cells compared to normal tissuecells. The distinguishably frequent cell division of cancer cells is thebasis for the effectiveness of existing cancer treatments, e.g.,irradiation therapy and the use of various chemo-therapeutic agents.Such treatments are based on the fact that cells undergoing division aremore sensitive to radiation and chemo-therapeutic agents thannon-dividing cells. Because tumors cells divide much more frequentlythan normal cells, it is possible, to a certain extent, to selectivelydamage or destroy tumor cells by radiation therapy and/or chemotherapy.The actual sensitivity of cells to radiation, therapeutic agents, etc.,is also dependent on specific characteristics of different types ofnormal or malignant cell types. Thus, unfortunately, the sensitivity oftumor cells is not sufficiently higher than that many types of normaltissues. This diminishes the ability to distinguish between tumor cellsand normal cells, and therefore, existing cancer treatments typicallycause significant damage to normal tissues, thus limiting thetherapeutic effectiveness of such treatments. Furthermore, theinevitable damage to other tissue renders treatments very traumatic tothe patients and, often, patients are unable to recover from a seeminglysuccessful treatment. Also, certain types of tumors are not sensitive atall to existing methods of treatment.

There are also other methods for destroying cells that do not rely onradiation therapy or chemotherapy alone. For example, ultrasonic andelectrical methods for destroying tumor cells can be used in addition toor instead of conventional treatments. Electric fields and currents havebeen used for medical purposes for many years. The most common is thegeneration of electric currents in a human or animal body by applicationof an electric field by means of a pair of conductive electrodes betweenwhich a potential difference is maintained. These electric currents areused either to exert their specific effects, i.e., to stimulateexcitable tissue, or to generate heat by flowing in the body since itacts as a resistor. Examples of the first type of application includethe following: cardiac defibrillators, peripheral nerve and musclestimulators, brain stimulators, etc. Currents are used for heating, forexample, in devices for tumor ablation, ablation of malfunctioningcardiac or brain tissue, cauterization, relaxation of muscle rheumaticpain and other pain, etc.

Another use of electric fields for medical purposes involves theutilization of high frequency oscillating fields transmitted from asource that emits an electric wave, such as an RF wave or a microwavesource that is directed at the part of the body that is of interest(i.e., target). In these instances, there is no electric energyconduction between the source and the body; but rather, the energy istransmitted to the body by radiation or induction. More specifically,the electric energy generated by the source reaches the vicinity of thebody via a conductor and is transmitted from it through air or someother electric insulating material to the human body.

In a conventional electrical method, electrical current is delivered toa region of the target tissue using electrodes that are placed incontact with the body of the patient. The applied electrical currentdestroys substantially all cells in the vicinity of the target tissue.Thus, this type of electrical method does not discriminate betweendifferent types of cells within the target tissue and results in thedestruction of both tumor cells and normal cells.

Electric fields that can be used in medical applications can thus beseparated generally into two different modes. In the first mode, theelectric fields are applied to the body or tissues by means ofconducting electrodes. These electric fields can be separated into twotypes, namely (1) steady fields or fields that change at relatively slowrates, and alternating fields of low frequencies that inducecorresponding electric currents in the body or tissues, and (2) highfrequency alternating fields (above 1 MHz) applied to the body by meansof the conducting electrodes. In the second mode, the electric fieldsare high frequency alternating fields applied to the body by means ofinsulated electrodes.

The first type of electric field is used, for example, to stimulatenerves and muscles, pace the heart, etc. In fact, such fields are usedin nature to propagate signals in nerve and muscle fibers, centralnervous system (CNS), heart, etc. The recording of such natural fieldsis the basis for the ECG, EEG, EMG, ERG, etc. The field strength inthese applications, assuming a medium of homogenous electric properties,is simply the voltage applied to the stimulating/recording electrodesdivided by the distance between them. These currents can be calculatedby Ohm's law and can have dangerous stimulatory effects on the heart andCNS and can result in potentially harmful ion concentration changes.Also, if the currents are strong enough, they can cause excessiveheating in the tissues. This heating can be calculated by the powerdissipated in the tissue (the product of the voltage and the current).

When such electric fields and currents are alternating, theirstimulatory power, on nerve, muscle, etc., is an inverse function of thefrequency. At frequencies above 1-10 KHz, the stimulation power of thefields approaches zero. This limitation is due to the fact thatexcitation induced by electric stimulation is normally mediated bymembrane potential changes, the rate of which is limited by the RCproperties (time constants on the order of 1 ms) of the membrane.

Regardless of the frequency, when such current inducing fields areapplied, they are associated with harmful side effects caused bycurrents. For example, one negative effect is the changes in ionicconcentration in the various “compartments” within the system, and theharmful products of the electrolysis taking place at the electrodes, orthe medium in which the tissues are imbedded. The changes in ionconcentrations occur whenever the system includes two or morecompartments between which the organism maintains ion concentrationdifferences. For example, for most tissues, [Ca⁺⁺] in the extracellularfluid is about 2×10⁻³ M, while in the cytoplasm of typical cells itsconcentration can be as low as 10⁻⁷ M. A current induced in such asystem by a pair of electrodes, flows in part from the extracellularfluid into the cells and out again into the extracellular medium. About2% of the current flowing into the cells is carried by the Ca⁺⁺ ions. Incontrast, because the concentration of intracellular Ca⁺⁺ is muchsmaller, only a negligible fraction of the currents that exits the cellsis carried by these ions. Thus, Ca⁺⁺ ions accumulate in the cells suchthat their concentrations in the cells increases, while theconcentration in the extracellular compartment may decrease. Theseeffects are observed for both DC and alternating currents (AC). The rateof accumulation of the ions depends on the current intensity ionmobilities, membrane ion conductance, etc. An increase in [Ca⁺⁺] isharmful to most cells and if sufficiently high will lead to thedestruction of the cells. Similar considerations apply to other ions. Inview of the above observations, long term current application to livingorganisms or tissues can result in significant damage. Another majorproblem that is associated with such electric fields, is due to theelectrolysis process that takes place at the electrode surfaces. Herecharges are transferred between the metal (electrons) and theelectrolytic solution (ions) such that charged active radicals areformed. These can cause significant damage to organic molecules,especially macromolecules and thus damage the living cells and tissues.

In contrast, when high frequency electric fields, above 1 MHz andusually in practice in the range of GHz, are induced in tissues by meansof insulated electrodes, the situation is quite different. These type offields generate only capacitive or displacement currents, rather thanthe conventional charge conducting currents. Under the effect of thistype of field, living tissues behave mostly according to theirdielectric properties rather than their electric conductive properties.Therefore, the dominant field effect is that due to dielectric lossesand heating. Thus, it is widely accepted that in practice, themeaningful effects of such fields on living organisms, are only thosedue to their heating effects, i.e., due to dielectric losses.

In U.S. Pat. No. 6,043,066 ('066) to Mangano, a method and device arepresented which enable discrete objects having a conducting inner core,surrounded by a dielectric membrane to be selectively inactivated byelectric fields via irreversible breakdown of their dielectric membrane.One potential application for this is in the selection and purging ofcertain biological cells in a suspension. According to the '066 patent,an electric field is applied for targeting selected cells to causebreakdown of the dielectric membranes of these tumor cells, whilepurportedly not adversely affecting other desired subpopulations ofcells. The cells are selected on the basis of intrinsic or induceddifferences in a characteristic electroporation threshold. Thedifferences in this threshold can depend upon a number of parameters,including the difference in cell size.

The method of the '066 patent is therefore based on the assumption thatthe electroporation threshold of tumor cells is sufficientlydistinguishable from that of normal cells because of differences in cellsize and differences in the dielectric properties of the cell membranes.Based upon this assumption, the larger size of many types of tumor cellsmakes these cells more susceptible to electroporation and thus, it maybe possible to selectively damage only the larger tumor cell membranesby applying an appropriate electric field. One disadvantage of thismethod is that the ability to discriminate is highly dependent upon celltype, for example, the size difference between normal cells and tumorcells is significant only in certain types of cells. Another drawback ofthis method is that the voltages which are applied can damage some ofthe normal cells and may not damage all of the tumor cells because thedifferences in size and membrane dielectric properties are largelystatistical and the actual cell geometries and dielectric properties canvary significantly.

What is needed in the art and has heretofore not been available is anapparatus for destroying dividing cells, wherein the apparatus betterdiscriminates between dividing cells, including single-celled organisms,and non-dividing cells and is capable of selectively destroying thedividing cells or organisms with substantially no effect on thenon-dividing cells or organisms.

SUMMARY

While they are dividing, cells are vulnerable to damage by AC electricfields that have specific frequency and field strength characteristics.The selective destruction of rapidly dividing cells can therefore beaccomplished by imposing an AC electric field in a target region forextended periods of time. Some of the cells that divide while the fieldis applied will be damaged, but the cells that do not divide will not beharmed. This selectively damages rapidly dividing cells like tumorcells, but does not harm normal cells that are not dividing. Since thevulnerability of the dividing cells is strongly related to the alignmentbetween the long axis of the dividing cells and the lines of force ofthe electric field, improved results are obtained by sequentiallyimposing the field in different directions.

A major use of the present apparatus is in the treatment of tumors byselective destruction of tumor cells with substantially no effect onnormal tissue cells, and thus, the exemplary apparatus is describedbelow in the context of selective destruction of tumor cells. It shouldbe appreciated however, that for purpose of the following description,the term “cell” may also refer to a single-celled organism (eubacteria,bacteria, yeast, protozoa), multi-celled organisms (fungi, algae, mold),and plants as or parts thereof that are not normally classified as“cells”. The exemplary apparatus enables selective destruction of cellsundergoing division in a way that is more effective and more accurate(e.g., more adaptable to be aimed at specific targets) than existingmethods. Further, the present apparatus causes minimal damage, if any,to normal tissue and, thus, reduces or eliminates many side-effectsassociated with existing selective destruction methods, such asradiation therapy and chemotherapy. The selective destruction ofdividing cells using the present apparatus does not depend on thesensitivity of the cells to chemical agents or radiation. Instead, theselective destruction of dividing cells is based on distinguishablegeometrical characteristics of cells undergoing division, in comparisonto non-dividing cells, regardless of the cell geometry of the type ofcells being treated.

According to one exemplary embodiment, cell geometry-dependent selectivedestruction of living tissue is performed by inducing a non-homogenouselectric field in the cells using an electronic apparatus.

It has been observed by the present inventor that, while different cellsin their non-dividing state may have different shapes, e.g., spherical,ellipsoidal, cylindrical, “pancake-like”, etc., the division process ofpractically all cells is characterized by development of a “cleavagefurrow” in late anaphase and telophase. This cleavage furrow is a slowconstriction of the cell membrane (between the two sets of daughterchromosomes) which appears microscopically as a growing cleft (e.g., agroove or notch) that gradually separates the cell into two new cells.During the division process, there is a transient period (telophase)during which the cell structure is basically that of two sub-cellsinterconnected by a narrow “bridge” formed of the cell material. Thedivision process is completed when the “bridge” between the twosub-cells is broken. The selective destruction of tumor cells using thepresent electronic apparatus utilizes this unique geometrical feature ofdividing cells.

When a cell or a group of cells are under natural conditions orenvironment, i.e., part of a living tissue, they are disposed surroundedby a conductive environment consisting mostly of an electrolyticinter-cellular fluid and other cells that are composed mostly of anelectrolytic intra-cellular liquid. When an electric field is induced inthe living tissue, by applying an electric potential across the tissue,an electric field is formed in the tissue and the specific distributionand configuration of the electric field lines defines the direction ofcharge displacement, or paths of electric currents in the tissue, ifcurrents are in fact induced in the tissue. The distribution andconfiguration of the electric field is dependent on various parametersof the tissue, including the geometry and the electric properties of thedifferent tissue components, and the relative conductivities, capacitiesand dielectric constants (that may be frequency dependent) of the tissuecomponents.

The electric current flow pattern for cells undergoing division is verydifferent and unique as compared to non-dividing cells. Such cellsincluding first and second sub-cells, namely an “original” cell and anewly formed cell, that are connected by a cytoplasm “bridge” or “neck”.The currents penetrate the first sub-cell through part of the membrane(“the current source pole”); however, they do not exit the firstsub-cell through a portion of its membrane closer to the opposite pole(“the current sink pole”). Instead, the lines of current flow convergeat the neck or cytoplasm bridge, whereby the density of the current flowlines is greatly increased. A corresponding, “mirror image”, processthat takes place in the second sub-cell, whereby the current flow linesdiverge to a lower density configuration as they depart from the bridge,and finally exit the second sub-cell from a part of its membrane closesto the current sink.

When a polarizable object is placed in a non-uniform converging ordiverging field, electric forces act on it and pull it towards thehigher density electric field lines. In the case of dividing cell,electric forces are exerted in the direction of the cytoplasm bridgebetween the two cells. Since all intercellular organelles andmacromolecules are polarizable, they are all force towards the bridgebetween the two cells. The field polarity is irrelevant to the directionof the force and, therefore, an alternating electric having specificproperties can be used to produce substantially the same effect. It willalso be appreciated that the concentrated and inhomogeneous electricfield present in or near the bridge or neck portion in itself exertsstrong forces on charges and natural dipoles and can lead to thedisruption of structures associated with these members.

The movement of the cellular organelles towards the bridge disrupts thecell structure and results in increased pressure in the vicinity of theconnecting bridge membrane. This pressure of the organelles on thebridge membrane is expected to break the bridge membrane and, thus, itis expected that the dividing cell will “explode” in response to thispressure. The ability to break the membrane and disrupt other cellstructures can be enhanced by applying a pulsating alternating electricfield that has a frequency from about 50 KHz to about 500 KHz. When thistype of electric field is applied to the tissue, the forces exerted onthe intercellular organelles have a “hammering” effect, whereby forcepulses (or beats) are applied to the organelles numerous times persecond, enhancing the movement of organelles of different sizes andmasses towards the bridge (or neck) portion from both of the sub-cells,thereby increasing the probability of breaking the cell membrane at thebridge portion. The forces exerted on the intracellular organelles alsoaffect the organelles themselves and may collapse or break theorganelles.

Advantageously, when non-dividing cells are subjected to these electricfields, there is no effect on the cells; however, the situation is muchdifferent when dividing cells are subjected to the present electricfields. Thus, the present electronic apparatus and the generatedelectric fields target dividing cells, such as tumors or the like, anddo not target non-dividing cells that is found around in healthy tissuesurrounding the target area. Furthermore, since the present apparatusutilizes insulated electrodes, the above mentioned negative effects,obtained when conductive electrodes are used, i.e., ion concentrationchanges in the cells and the formation of harmful agents byelectrolysis, do not occur with the present apparatus. This is because,in general, no actual transfer of charges takes place between theelectrodes and the medium, and there is no charge flow in the mediumwhere the currents are capacitive.

It should be appreciated that the present electronic apparatus can alsobe used in applications other than treatment of tumors in the livingbody. In fact, the selective destruction utilizing the present apparatuscan be used in conjunction with any organism that proliferates bydivision, for example, tissue cultures, microorganisms, such asbacteria, mycoplasma, protozoa, fungi, algae, plant cells, etc. Suchorganisms divide by the formation of a groove or cleft as describedabove. As the groove or cleft deepens, a narrow bridge is formed betweenthe two parts of the organism, similar to the bridge formed between thesub-cells of dividing animal cells. Since such organisms are covered bya membrane having a relatively low electric conductivity, similar to ananimal cell membrane described above, the electric field lines in adividing organism converge at the bridge connecting the two parts of thedividing organism. The converging field lines result in electric forcesthat displace polarizable elements and charges within the dividingorganism.

One aspect of the invention relates to a method of selectivelydestroying or inhibiting the growth of bacteria located within a targetregion of a patient. This method includes the steps of administering anantibiotic against the bacteria, so that a therapeutically effectivedose arrives in the target region and capacitively coupling an ACelectric field into the target region of the patient while thetherapeutically effective dose is present at the target region, whereinthe electric field has frequency characteristics that correspond to avulnerability of the bacteria, and wherein the electric field is strongenough to damage, during cell division, a significant portion of thebacteria whose long axis is generally aligned with the lines of force ofthe electric field. The strength of the electric field in at least aportion of the target region is between 2 V/cm and 100 V/cm, and theelectric field leaves non-dividing cells located within the targetregion substantially unharmed. The coupling step is repeated until atherapeutically significant portion of the bacteria die.

Another aspect of the invention relates to a method of selectivelydestroying or inhibiting the growth of bacteria located within a targetregion. This method includes the steps of capacitively coupling an ACelectric field into the target region. The frequency of the electricfield is between 5 MHz and 20 MHz, and the strength of the electricfield in at least a portion of the target region is between 2 V/cm and100 V/cm. The electric field has frequency characteristics thatcorrespond to a vulnerability of the bacteria and the electric field isstrong enough to damage, during cell division, a significant portion ofthe bacteria whose long axis is generally aligned with the lines offorce of the electric field, and leave non-dividing cells located withinthe target region substantially unharmed. The coupling step is repeateduntil a therapeutically significant portion of the bacteria die.

Another aspect of the invention relates to an apparatus for selectivelydestroying or inhibiting the growth of bacteria located within a targetregion of a patient. This apparatus includes a first pair of insulatedelectrodes, and each of the electrodes has a surface configured tofacilitate capacitive coupling of an electric field into the patient'sbody. The apparatus also includes an AC voltage source operativelyconnected to the electrodes. The AC voltage source and the electrodesare configured so that, when the electrodes are placed against thepatient's body and the AC voltage source is activated, an AC electricfield is capacitively coupled into the target region of the patient viathe electrodes. The frequency of the electric field is between 5 MHz and20 MHz, and the strength of the electric field in at least a portion ofthe target region is between 2 V/cm and 100 V/cm. The imposed electricfield has frequency characteristics that correspond to a vulnerabilityof the bacteria, and is strong enough to damage, during cell division, asignificant portion of the bacteria whose long axis is generally alignedwith the lines of force of the electric field. The electric field alsoleaves non-dividing cells located within the target region substantiallyunharmed.

The above, and other objects, features and advantages of the presentapparatus will become apparent from the following description read inconjunction with the accompanying drawings, in which like referencenumerals designate the same elements.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1E are simplified, schematic, cross-sectional, illustrations ofvarious stages of a cell division process;

FIGS. 2A and 2B are schematic illustrations of a non-dividing cell beingsubjected to an electric field;

FIGS. 3A, 3B and 3C are schematic illustrations of a dividing cell beingsubjected to an electric field according to one exemplary embodiment,resulting in destruction of the cell (FIG. 3C) in accordance with oneexemplary embodiment;

FIG. 4 is a schematic illustration of a dividing cell at one stage beingsubject to an electric field;

FIG. 5 is a schematic block diagram of an apparatus for applying anelectric according to one exemplary embodiment for selectivelydestroying cells;

FIG. 6 is a simplified schematic diagram of an equivalent electriccircuit of insulated electrodes of the apparatus of FIG. 5;

FIG. 7 is a cross-sectional illustration of a skin patch incorporatingthe apparatus of FIG. 5 and for placement on a skin surface for treatinga tumor or the like;

FIG. 8 is a cross-sectional illustration of the insulated electrodesimplanted within the body for treating a tumor or the like;

FIG. 9 is a cross-sectional illustration of the insulated electrodesimplanted within the body for treating a tumor or the like;

FIGS. 10A-10D are cross-sectional illustrations of various constructionsof the insulated electrodes of the apparatus of FIG. 5;

FIG. 11 is a front elevational view in partial cross-section of twoinsulated electrodes being arranged about a human torso for treatment ofa tumor container within the body, e.g., a tumor associated with lungcancer;

FIGS. 12A-12C are cross-sectional illustrations of various insulatedelectrodes with and without protective members formed as a part of theconstruction thereof;

FIG. 13 is a schematic diagram of insulated electrodes that are arrangedfor focusing the electric field at a desired target while leaving otherareas in low field density (i.e., protected areas);

FIG. 14 is a cross-sectional view of insulated electrodes incorporatedinto a hat according to a first embodiment for placement on a head fortreating an intra-cranial tumor or the like;

FIG. 15 is a partial section of a hat according to an exemplaryembodiment having a recessed section for receiving one or more insulatedelectrodes;

FIG. 16 is a cross-sectional view of the hat of FIG. 15 placed on a headand illustrating a biasing mechanism for applying a force to theinsulated electrode to ensure the insulated electrode remains in contactagainst the head;

FIG. 17 is a cross-sectional top view of an article of clothing havingthe insulated electrodes incorporated therein for treating a tumor orthe like;

FIG. 18 is a cross-sectional view of a section of the article ofclothing of FIG. 17 illustrating a biasing mechanism for biasing theinsulated electrode in direction to ensure the insulated electrode isplaced proximate to a skin surface where treatment is desired;

FIG. 19 is a cross-sectional view of a probe according to one embodimentfor being disposed internally within the body for treating a tumor orthe like;

FIG. 20 is an elevational view of an unwrapped collar according to oneexemplary embodiment for placement around a neck for treating a tumor orthe like in this area when the collar is wrapped around the neck;

FIG. 21 is a cross-sectional view of two insulated electrodes withconductive gel members being arranged about a body, with the electricfield lines being shown;

FIG. 22 is a cross-sectional view of the arrangement of FIG. 21illustrating a point of insulation breakdown in one insulated electrode;

FIG. 23 is a cross-sectional view of an arrangement of at least twoinsulated electrodes with conductive gel members being arranged about abody for treatment of a tumor or the like, wherein each conductive gelmember has a feature for minimizing the effects of an insulationbreakdown in the insulated electrode;

FIG. 24 is a cross-sectional view of another arrangement of at least twoinsulated electrodes with conductive gel members being arranged about abody for treatment of a tumor or the like, wherein a conductive memberis disposed within the body near the tumor to create a region ofincreased field density;

FIG. 25 is a cross-sectional view of an arrangement of two insulatedelectrodes of varying sizes disposed relative to a body; and

FIG. 26 is a cross-sectional view of an arrangement of at least twoinsulated electrodes with conductive gel members being arranged about abody for treatment of a tumor or the like, wherein each conductive gelmember has a feature for minimizing the effects of an insulationbreakdown in the insulated electrode.

FIGS. 27A-C show a configuration of electrodes that facilitates theapplication of an electric field in different directions.

FIG. 28 shows a three-dimensional arrangement of electrodes about a bodypart that facilitates the application of an electric field in differentdirections.

FIGS. 29A and 29B are graphs of the efficiency of the cell destructionprocess as a function of field strength for melanoma and glioma cells,respectively.

FIGS. 30A and 30B are graphs that show how the cell destructionefficiency is a function of the frequency of the applied field formelanoma and glioma cells, respectively.

FIG. 31A is a graphical representation of the sequential application ofa plurality of frequencies in a plurality of directions.

FIG. 31B is a graphical representation of the sequential application ofa sweeping frequency in a plurality of directions.

FIG. 32A depicts the construction of the electrodes used in anexperiment on bacteria.

FIG. 32B depicts a test chamber used in an experiment on bacteria.

FIG. 32C depicts a setup that was used to induce fields in the testchamber.

FIGS. 33A and 33B show the effect of treating bacteria with electricfields at different frequencies.

FIG. 34 shows the effect of treating bacteria with electric fields atdifferent field strengths.

FIGS. 35A and 35B show the effect of treating bacteria with electricfields at different switching rates.

FIG. 36 shows the results of this repeated exposure test of P.aeruginosa to electric fields.

FIG. 37 shows the electric field distribution in and around a bacteria.

FIGS. 38A and 38B show the magnitude of the forces acting on dipolesinside dividing bacteria.

FIG. 39 shows the results of an in vivo experiment.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Reference is made to FIGS. 1A-1E which schematically illustrate variousstages of a cell division process. FIG. 1A illustrates a cell 10 at itsnormal geometry, which can be generally spherical (as illustrated in thedrawings), ellipsoidal, cylindrical, “pancake-like” or any other cellgeometry, as is known in the art. FIGS. 1B-1D illustrate cell 10 duringdifferent stages of its division process, which results in the formationof two new cells 18 and 20, shown in FIG. 1E.

As shown in FIGS. 1B-1D, the division process of cell 10 ischaracterized by a slowly growing cleft 12 which gradually separatescell 10 into two units, namely sub-cells 14 and 16, which eventuallyevolve into new cells 18 and 20 (FIG. 1E). A shown specifically in FIG.1D, the division process is characterized by a transient period duringwhich the structure of cell 10 is basically that of the two sub-cells 14and 16 interconnected by a narrow “bridge” 22 containing cell material(cytoplasm surrounded by cell membrane).

Reference is now made to FIGS. 2A and 2B, which schematically illustratenon-dividing cell 10 being subjected to an electric field produced byapplying an alternating electric potential, at a relatively lowfrequency and at a relatively high frequency, respectively. Cell 10includes intracellular organelles, e.g., a nucleus 30. Alternatingelectric potential is applied across electrodes 28 and 32 that can beattached externally to a patient at a predetermined region, e.g., in thevicinity of the tumor being treated. When cell 10 is under naturalconditions, i.e., part of a living tissue, it is disposed in aconductive environment (hereinafter referred to as a “volume conductor”)consisting mostly of electrolytic inter-cellular liquid. When anelectric potential is applied across electrodes 28 and 32, some of thefield lines of the resultant electric field (or the current induced inthe tissue in response to the electric field) penetrate the cell 10,while the rest of the field lines (or induced current) flow in thesurrounding medium. The specific distribution of the electric fieldlines, which is substantially consistent with the direction of currentflow in this instance, depends on the geometry and the electricproperties of the system components, e.g., the relative conductivitiesand dielectric constants of the system components, that can be frequencydependent. For low frequencies, e.g., frequencies lower than 10 KHz, theconductance properties of the components completely dominate the currentflow and the field distribution, and the field distribution is generallyas depicted in FIG. 2A. At higher frequencies, e.g., at frequencies ofbetween 10 KHz and 1 MHz, the dielectric properties of the componentsbecomes more significant and eventually dominate the field distribution,resulting in field distribution lines as depicted generally in FIG. 2B.

For constant (i.e., DC) electric fields or relatively low frequencyalternating electric fields, for example, frequencies under 10 KHz, thedielectric properties of the various components are not significant indetermining and computing the field distribution. Therefore, as a firstapproximation, with regard to the electric field distribution, thesystem can be reasonably represented by the relative impedances of itsvarious components. Using this approximation, the intercellular (i.e.,extracellular) fluid and the intracellular fluid each has a relativelylow impedance, while the cell membrane 11 has a relatively highimpedance. Thus, under low frequency conditions, only a fraction of theelectric field lines (or currents induced by the electric field)penetrate membrane 11 of the cell 10. At relatively high frequencies(e.g., 10 KHz-1 MHz), in contrast, the impedance of membrane 11 relativeto the intercellular and intracellular fluids decreases, and thus, thefraction of currents penetrating the cells increases significantly. Itshould be noted that at very high frequencies, i.e., above 1 MHz, themembrane capacitance can short the membrane resistance and, therefore,the total membrane resistance can become negligible.

In any of the embodiments described above, the electric field lines (orinduced currents) penetrate cell 10 from a portion of the membrane 11closest to one of the electrodes generating the current, e.g., closestto positive electrode 28 (also referred to herein as “source”). Thecurrent flow pattern across cell 10 is generally uniform because, underthe above approximation, the field induced inside the cell issubstantially homogeneous. The currents exit cell 10 through a portionof membrane 11 closest to the opposite electrode, e.g., negativeelectrode 32 (also referred to herein as “sink”).

The distinction between field lines and current flow can depend on anumber of factors, for example, on the frequency of the applied electricpotential and on whether electrodes 28 and 32 are electricallyinsulated. For insulated electrodes applying a DC or low frequencyalternating voltage, there is practically no current flow along thelines of the electric field. At higher frequencies, the displacementcurrents are induced in the tissue due to charging and discharging ofthe electrode insulation and the cell membranes (which act as capacitorsto a certain extent), and such currents follow the lines of the electricfield. Fields generated by non-insulated electrodes, in contrast, alwaysgenerate some form of current flow, specifically, DC or low frequencyalternating fields generate conductive current flow along the fieldlines, and high frequency alternating fields generate both conductionand displacement currents along the field lines. It should beappreciated, however, that movement of polarizable intracellularorganelles according to the present invention (as described below) isnot dependent on actual flow of current and, therefore, both insulatedand non-insulated electrodes can be used efficiently. Advantages ofinsulated electrodes include lower power consumption, less heating ofthe treated regions, and improved patient safety.

According to one exemplary embodiment of the present invention, theelectric fields that are used are alternating fields having frequenciesthat are in the range from about 50 KHz to about 500 KHz, and preferablyfrom about 100 KHz to about 300 KHz. For ease of discussion, these typeof electric fields are also referred to below as “TC fields”, which isan abbreviation of “Tumor Curing electric fields”, since these electricfields fall into an intermediate category (between high and lowfrequency ranges) that have bio-effective field properties while havingno meaningful stimulatory and thermal effects. These frequencies aresufficiently low so that the system behavior is determined by thesystem's Ohmic (conductive) properties but sufficiently high enough notto have any stimulation effect on excitable tissues. Such a systemconsists of two types of elements, namely, the intercellular, orextracellular fluid, or medium and the individual cells. Theintercellular fluid is mostly an electrolyte with a specific resistanceof about 40-100 Ohm*cm. As mentioned above, the cells are characterizedby three elements, namely (1) a thin, highly electric resistive membranethat coats the cell; (2) internal cytoplasm that is mostly anelectrolyte that contains numerous macromolecules and micro-organelles,including the nucleus; and (3) membranes, similar in their electricproperties to the cell membrane, cover the micro-organelles.

When this type of system is subjected to the present TC fields (e.g.,alternating electric fields in the frequency range of 100 KHz-300 KHz)most of the lines of the electric field and currents tend away from thecells because of the high resistive cell membrane and therefore thelines remain in the extracellular conductive medium. In the aboverecited frequency range, the actual fraction of electric field orcurrents that penetrates the cells is a strong function of thefrequency.

FIG. 2 schematically depicts the resulting field distribution in thesystem. As illustrated, the lines of force, which also depict the linesof potential current flow across the cell volume mostly in parallel withthe undistorted lines of force (the main direction of the electricfield). In other words, the field inside the cells is mostlyhomogeneous. In practice, the fraction of the field or current thatpenetrates the cells is determined by the cell membrane impedance valuerelative to that of the extracellular fluid. Since the equivalentelectric circuit of the cell membrane is that of a resistor andcapacitor in parallel, the impedance is a function of the frequency. Thehigher the frequency, the lower the impedance, the larger the fractionof penetrating current and the smaller the field distortion (RotshenkerS. & Y. Palti, Changes in fraction of current penetrating an axon as afunction of duration of stimulating pulse, J. Theor. Biol. 41; 401-407(1973).

As previously mentioned, when cells are subjected to relatively weakelectric fields and currents that alternate at high frequencies, such asthe present TC fields having a frequency in the range of 50 KHz-500 KHz,they have no effect on the non-dividing cells. While the present TCfields have no detectable effect on such systems, the situation becomesdifferent in the presence of dividing cells.

Reference is now made to FIGS. 3A-3C which schematically illustrate theelectric current flow pattern in cell 10 during its division process,under the influence of alternating fields (TC fields) in the frequencyrange from about 100 KHz to about 300 KHz in accordance with oneexemplary embodiment. The field lines or induced currents penetrate cell10 through a part of the membrane of sub-cell 16 closer to electrode 28.However, they do not exit through the cytoplasm bridge 22 that connectssub-cell 16 with the newly formed yet still attached sub-cell 14, orthrough a part of the membrane in the vicinity of the bridge 22.Instead, the electric field or current flow lines—that are relativelywidely separated in sub-cell 16—converge as they approach bridge 22(also referred to as “neck” 22) and, thus, the current/field linedensity within neck 22 is increased dramatically. A “mirror image”process takes place in sub-cell 14, whereby the converging field linesin bridge 22 diverge as they approach the exit region of sub-cell 14.

It should be appreciated by persons skilled in the art that homogeneouselectric fields do not exert a force on electrically neutral objects,i.e., objects having substantially zero net charge, although suchobjects can become polarized. However, under a non-uniform, convergingelectric field, as shown in FIGS. 3A-3C, electric forces are exerted onpolarized objects, moving them in the direction of the higher densityelectric field lines. It will be appreciated that the concentratedelectric field that is present in the neck or bridge area in itselfexerts strong forces on charges and natural dipoles and can disruptstructures that are associated therewith. One will understand thatsimilar net forces act on charges in an alternating field, again in thedirection of the field of higher intensity.

In the configuration of FIGS. 3A and 3B, the direction of movement ofpolarized and charged objects is towards the higher density electricfield lines, i.e., towards the cytoplasm bridge 22 between sub-cells 14and 16. It is known in the art that all intracellular organelles, forexample, nuclei 24 and 26 of sub-cells 14 and 16, respectively, arepolarizable and, thus, such intracellular organelles are electricallyforced in the direction of the bridge 22. Since the movement is alwaysfrom lower density currents to the higher density currents, regardlessof the field polarity, the forces applied by the alternating electricfield to organelles, such as nuclei 24 and 26, are always in thedirection of bridge 22. A comprehensive description of such forces andthe resulting movement of macromolecules of intracellular organelles, aphenomenon referred to as “dielectrophoresis” is described extensivelyin literature, e.g., in C. L. Asbury & G. van den Engh, Biophys. J. 74,1024-1030, 1998, the disclosure of which is hereby incorporated byreference in its entirety.

The movement of the organelles 24 and 26 towards the bridge 22 disruptsthe structure of the dividing cell, change the concentration of thevarious cell constituents and, eventually, the pressure of theconverging organelles on bridge membrane 22 results in the breakage ofcell membrane 11 at the vicinity of the bridge 22, as shownschematically in FIG. 3C. The ability to break membrane 11 at bridge 22and to otherwise disrupt the cell structure and organization can beenhanced by applying a pulsating AC electric field, rather than a steadyAC field. When a pulsating field is applied, the forces acting onorganelles 24 and 26 have a “hammering” effect, whereby pulsed forcesbeat on the intracellular organelles towards the neck 22 from bothsub-cells 14 and 16, thereby increasing the probability of breaking cellmembrane 11 in the vicinity of neck 22.

A very important element, which is very susceptible to the specialfields that develop within the dividing cells is the microtubule spindlethat plays a major role in the division process. In FIG. 4, a dividingcell 10 is illustrated, at an earlier stage as compared to FIGS. 3A and3B, under the influence of external TC fields (e.g., alternating fieldsin the frequency range of about 100 KHz to about 300 KHz), generallyindicated as lines 100, with a corresponding spindle mechanism generallyindicated at 120. The lines 120 are microtubules that are known to havea very strong dipole moment. This strong polarization makes the tubules,as well as other polar macromolecules and especially those that have aspecific orientation within the cells or its surrounding, susceptible toelectric fields. Their positive charges are located at the twocentrioles while two sets of negative poles are at the center of thedividing cell and the other pair is at the points of attachment of themicrotubules to the cell membrane, generally indicated at 130. Thisstructure forms sets of double dipoles and therefore they aresusceptible to fields of different directions. It will be understoodthat the effect of the TC fields on the dipoles does not depend on theformation of the bridge (neck) and thus, the dipoles are influenced bythe TC fields prior to the formation of the bridge (neck).

Since the present apparatus (as will be described in greater detailbelow) utilizes insulated electrodes, the above-mentioned negativeeffects obtained when conductive electrodes are used, i.e., ionconcentration changes in the cells and the formation of harmful agentsby electrolysis, do not occur when the present apparatus is used. Thisis because, in general, no actual transfer of charges takes placebetween the electrodes and the medium and there is no charge flow in themedium where the currents are capacitive, i.e., are expressed only asrotation of charges, etc.

Turning now to FIG. 5, the TC fields described above that have beenfound to advantageously destroy tumor cells are generated by anelectronic apparatus 200. FIG. 5 is a simple schematic diagram of theelectronic apparatus 200 illustrating the major components thereof. Theelectronic apparatus 200 generates the desired electric signals (TCsignals) in the shape of waveforms or trains of pulses. The apparatus200 includes a generator 210 and a pair of conductive leads 220 that areattached at one end thereof to the generator 210. The opposite ends ofthe leads 220 are connected to insulated conductors 230 that areactivated by the electric signals (e.g., waveforms). The insulatedconductors 230 are also referred to hereinafter as isolects 230.Optionally and according to another exemplary embodiment, the apparatus200 includes a temperature sensor 240 and a control box 250 which areboth added to control the amplitude of the electric field generated soas not to generate excessive heating in the area that is treated.

The generator 210 generates an alternating voltage waveform atfrequencies in the range from about 50 KHz to about 500 KHz (preferablyfrom about 100 KHz to about 300 KHz) (i.e., the TC fields). The requiredvoltages are such that the electric field intensity in the tissue to betreated is in the range of about 0.1 V/cm to about 10 V/cm. To achievethis field, the actual potential difference between the two conductorsin the isolects 230 is determined by the relative impedances of thesystem components, as described below.

When the control box 250 is included, it controls the output of thegenerator 210 so that it will remain constant at the value preset by theuser or the control box 250 sets the output at the maximal value thatdoes not cause excessive heating, or the control box 250 issues awarning or the like when the temperature (sensed by temperature sensor240) exceeds a preset limit.

The leads 220 are standard isolated conductors with a flexible metalshield, preferably grounded so that it prevents the spread of theelectric field generated by the leads 220. The isolects 230 havespecific shapes and positioning so as to generate an electric field ofthe desired configuration, direction, and intensity at the target volumeand only there so as to focus the treatment.

The specifications of the apparatus 200 as a whole and its individualcomponents are largely influenced by the fact that at the frequency ofthe present TC fields (50 KHz-500 KHz), living systems behave accordingto their “Ohmic”, rather than their dielectric properties. The onlyelements in the apparatus 200 that behave differently are the insulatorsof the isolects 230 (see FIGS. 7-9). The isolects 200 consist of aconductor in contact with a dielectric that is in contact with theconductive tissue thus forming a capacitor.

The details of the construction of the isolects 230 is based on theirelectric behavior that can be understood from their simplified electriccircuit when in contact with tissue as generally illustrated in FIG. 6.In the illustrated arrangement, the potential drop or the electric fielddistribution between the different components is determined by theirrelative electric impedance, i.e., the fraction of the field on eachcomponent is given by the value of its impedance divided by the totalcircuit impedance. For example, the potential drop on element ΔV_(A)=A/(A+B+C+D+E). Thus, for DC or low frequency AC, practically allthe potential drop is on the capacitor (that acts as an insulator). Forrelatively very high frequencies, the capacitor practically is a shortand therefore, practically all the field is distributed in the tissues.At the frequencies of the present TC fields (e.g., 50 KHz to 500 KHz),which are intermediate frequencies, the impedance of the capacitance ofthe capacitors is dominant and determines the field distribution.Therefore, in order to increase the effective voltage drop across thetissues (field intensity), the impedance of the capacitors is to bedecreased (i.e., increase their capacitance). This can be achieved byincreasing the effective area of the “plates” of the capacitor, decreasethe thickness of the dielectric or use a dielectric with high dielectricconstant.

In order to optimize the field distribution, the isolects 230 areconfigured differently depending upon the application in which theisolects 230 are to be used. There are two principle modes for applyingthe present electric fields (TC fields). First, the TC fields can beapplied by external isolects and second, the TC fields can be applied byinternal isolects.

Electric fields (TC fields) that are applied by external isolects can beof a local type or widely distributed type. The first type includes, forexample, the treatment of skin tumors and treatment of lesions close tothe skin surface. FIG. 7 illustrates an exemplary embodiment where theisolects 230 are incorporated in a skin patch 300. The skin patch 300can be a self-adhesive flexible patch with one or more pairs of isolects230. The patch 300 includes internal insulation 310 (formed of adielectric material) and the external insulation 260 and is applied toskin surface 301 that contains a tumor 303 either on the skin surface301 or slightly below the skin surface 301. Tissue is generallyindicated at 305. To prevent the potential drop across the internalinsulation 310 to dominate the system, the internal insulation 310 musthave a relatively high capacity. This can be achieved by a large surfacearea; however, this may not be desired as it will result in the spreadof the field over a large area (e.g., an area larger than required totreat the tumor). Alternatively, the internal insulation 310 can be madevery thin and/or the internal insulation 310 can be of a high dielectricconstant. As the skin resistance between the electrodes (labeled as Aand E in FIG. 6) is normally significantly higher than that of thetissue (labeled as C in FIG. 6) underneath it (1-10 KΩ vs. 0.1-1 KΩ),most of the potential drop beyond the isolects occurs there. Toaccommodate for these impedances (Z), the characteristics of theinternal insulation 310 (labeled as B and D in FIG. 6) should be suchthat they have impedance preferably under 100 KΩ at the frequencies ofthe present TC fields (e.g., 50 KHz to 500 KHz). For example, if it isdesired for the impedance to be about 10 K Ohms or less, such that over1% of the applied voltage falls on the tissues, for isolects with asurface area of 10 mm², at frequencies of 200 KHz, the capacity shouldbe on the order of 10⁻¹⁰ F., which means that using standard insulationswith a dielectric constant of 2-3, the thickness of the insulating layer310 should be about 50-100 microns. An internal field 10 times strongerwould be obtained with insulators with a dielectric constant of about20-50.

Using an insulating material with a high dielectric constant increasesthe capacitance of the electrodes, which results in a reduction of theelectrodes' impedance to the AC signal that is applied by the generator1 (shown in FIG. 5). Because the electrodes A, E are wired in serieswith the target tissue C, as shown in FIG. 6, this reduction inimpedance reduces the voltage drop in the electrodes, so that a largerportion of the applied AC voltage appears across the tissue C. Since alarger portion of the voltage appears across the tissue, the voltagethat is being applied by the generator 1 can be advantageously loweredfor a given field strength in the tissue.

The desired field strength in the tissue being treated is preferablybetween about 0.1 V/cm and about 10 V/cm, and more preferably betweenabout 2 V/cm and 3 V/cm or between about 1 V/cm and about 5 V/cm. If thedielectric constant used in the electrode is sufficiently high, theimpedance of the electrodes A, E drops down to the same order ofmagnitude as the series combination of the skin and tissue B, C, D. Oneexample of a suitable material with an extremely high dielectricconstant is CaCu₃Ti₄O₁₂, which has a dielectric constant of about 11,000(measured at 100 kHz). When the dielectric constant is this high, usefulfields can be obtained using a generator voltage that is on the order ofa few tens of Volts.

Since the thin insulating layer can be very vulnerable, etc., theinsulation can be replaced by very high dielectric constant insulatingmaterials, such as titanium dioxide (e.g., rutile), the dielectricconstant can reach values of about 200. There a number of differentmaterials that are suitable for use in the intended application and havehigh dielectric constants. For example, some materials include: lithiumniobate (LiNbO₃), which is a ferroelectric crystal and has a number ofapplications in optical, pyroelectric and piezoelectric devices; yttriumiron garnet (YIG) is a ferromagnetic crystal and magneto-opticaldevices, e.g., optical isolator can be realized from this material;barium titanate (BaTiO₃) is a ferromagnetic crystal with a largeelectro-optic effect; potassium tantalate (KTaO₃) which is a dielectriccrystal (ferroelectric at low temperature) and has very low microwaveloss and tunability of dielectric constant at low temperature; andlithium tantalate (LiTaO₃) which is a ferroelectric crystal with similarproperties as lithium niobate and has utility in electro-optical,pyroelectric and piezoelectric devices. Insulator ceramics with highdielectric constants may also be used, such as a ceramic made of acombination of Lead Magnesium Niobate and Lead Titanate. It will beunderstood that the aforementioned exemplary materials can be used incombination with the present device where it is desired to use amaterial having a high dielectric constant.

One must also consider another factor that affects the effectivecapacity of the isolects 230, namely the presence of air between theisolects 230 and the skin. Such presence, which is not easy to prevent,introduces a layer of an insulator with a dielectric constant of 1.0, afactor that significantly lowers the effective capacity of the isolects230 and neutralizes the advantages of the titanium dioxide (rutile),etc. To overcome this problem, the isolects 230 can be shaped so as toconform with the body structure and/or (2) an intervening filler 270 (asillustrated in FIG. 10C), such as a gel, that has high conductance and ahigh effective dielectric constant, can be added to the structure. Theshaping can be pre-structured (see FIG. 10A) or the system can be madesufficiently flexible so that shaping of the isolects 230 is readilyachievable. The gel can be contained in place by having an elevated rimas depicted in FIGS. 10C and 10C′. The gel can be made of hydrogels,gelatins, agar, etc., and can have salts dissolved in it to increase itsconductivity. FIGS. 10A-10C′ illustrate various exemplary configurationsfor the isolects 230. The exact thickness of the gel is not important solong as it is of sufficient thickness that the gel layer does not dryout during the treatment. In one exemplary embodiment, the thickness ofthe gel is about 0.5 mm to about 2 mm. Preferably, the gel has highconductivity, is tacky, and is biocompatible for extended periods oftime. One suitable gel is AG603 Hydrogel, which is available from AmGelTechnologies, 1667 S. Mission Road, Fallbrook, Calif. 92028-4115, USA.

In order to achieve the desirable features of the isolects 230, thedielectric coating of each should be very thin, for example from between1-50 microns. Since the coating is so thin, the isolects 230 can easilybe damaged mechanically or undergo dielectric breakdown. This problemcan be overcome by adding a protective feature to the isolect'sstructure so as to provide desired protection from such damage. Forexample, the isolect 230 can be coated, for example, with a relativelyloose net 340 that prevents access to the surface but has only a minoreffect on the effective surface area of the isolect 230 (i.e., thecapacity of the isolects 230 (cross section presented in FIG. 12B). Theloose net 340 does not affect the capacity and ensures good contact withthe skin, etc. The loose net 340 can be formed of a number of differentmaterials; however, in one exemplary embodiment, the net 340 is formedof nylon, polyester, cotton, etc. Alternatively, a very thin conductivecoating 350 can be applied to the dielectric portion (insulating layer)of the isolect 230. One exemplary conductive coating is formed of ametal and more particularly of gold. The thickness of the coating 350depends upon the particular application and also on the type of materialused to form the coating 350; however, when gold is used, the coatinghas a thickness from about 0.1 micron to about 0.1 mm. Furthermore, therim illustrated in FIG. 10 can also provide some mechanical protection.

However, the capacity is not the only factor to be considered. Thefollowing two factors also influence how the isolects 230 areconstructed. The dielectric strength of the internal insulating layer310 and the dielectric losses that occur when it is subjected to the TCfield, i.e., the amount of heat generated. The dielectric strength ofthe internal insulation 310 determines at what field intensity theinsulation will be “shorted” and cease to act as an intact insulation.Typically, insulators, such as plastics, have dielectric strength valuesof about 100V per micron or more. As a high dielectric constant reducesthe field within the internal insulator 310, a combination of a highdielectric constant and a high dielectric strength gives a significantadvantage. This can be achieved by using a single material that has thedesired properties or it can be achieved by a double layer with thecorrect parameters and thickness. In addition, to further decreasing thepossibility that the insulating layer 310 will fail, all sharp edges ofthe insulating layer 310 should be eliminated as by rounding thecorners, etc., as illustrated in FIG. 10D using conventional techniques.

FIGS. 8 and 9 illustrate a second type of treatment using the isolects230, namely electric field generation by internal isolects 230. A bodyto which the isolects 230 are implanted is generally indicated at 311and includes a skin surface 313 and a tumor 315. In this embodiment, theisolects 230 can have the shape of plates, wires or other shapes thatcan be inserted subcutaneously or a deeper location within the body 311so as to generate an appropriate field at the target area (tumor 315).

It will also be appreciated that the mode of isolects application is notrestricted to the above descriptions. In the case of tumors in internalorgans, for example, liver, lung, etc., the distance between each memberof the pair of isolects 230 can be large. The pairs can even bypositioned opposite sides of a torso 410, as illustrated in FIG. 11. Thearrangement of the isolects 230 in FIG. 11 is particularly useful fortreating a tumor 415 associated with lung cancer or gastro-intestinaltumors. In this embodiment, the electric fields (TC fields) spread in awide fraction of the body.

In order to avoid overheating of the treated tissues, a selection ofmaterials and field parameters is needed. The isolects insulatingmaterial should have minimal dielectric losses at the frequency rangesto be used during the treatment process. This factor can be taken intoconsideration when choosing the particular frequencies for thetreatment. The direct heating of the tissues will most likely bedominated by the heating due to current flow (given by the I*R product).In addition, the isolect (insulated electrode) 230 and its surroundingsshould be made of materials that facilitate heat losses and its generalstructure should also facilitate head losses, i.e., minimal structuresthat block heat dissipation to the surroundings (air) as well as highheat conductivity. Using larger electrodes also minimizes the localsensation of heating, since it spreads the energy that is beingtransferred into the patient over a larger surface area. Preferably, theheating is minimized to the point where the patient's skin temperaturenever exceeds about 39° C.

Another way to reduce heating is to apply the field to the tissue beingtreated intermittently, by applying a field with a duty cycle betweenabout 20% and about 50% instead of using a continuous field. Forexample, to achieve a duty cycle of 33%, the field would be repetitivelyswitched on for one second, then switched off for two seconds.Preliminary experiments have shown that the efficacy of treatment usinga field with a 33% duty cycle is roughly the same as for a field with aduty cycle of 100%. In alternative embodiments, the field could beswitched on for one hour then switched off for one hour to achieve aduty cycle of 50%. Of course, switching at a rate of once per hour wouldnot help minimize short-term heating. On the other hand, it couldprovide the patient with a welcome break from treatment.

The effectiveness of the treatment can be enhanced by an arrangement ofisolects 230 that focuses the field at the desired target while leavingother sensitive areas in low field density (i.e., protected areas). Theproper placement of the isolects 230 over the body can be maintainedusing any number of different techniques, including using a suitablepiece of clothing that keeps the isolects at the appropriate positions.FIG. 13 illustrates such an arrangement in which an area labeled as “P”represents a protected area. The lines of field force do not penetratethis protected area and the field there is much smaller than near theisolects 230 where target areas can be located and treated well. Incontrast, the field intensity near the four poles is very high.

The following Example serves to illustrate an exemplary application ofthe present apparatus and application of TC fields; however, thisExample is not limiting and does not limit the scope of the presentinvention in any way.

Example 1

To demonstrate the effectiveness of electric fields having the abovedescribed properties (e.g., frequencies between 50 KHz and 500 KHz) indestroying tumor cells, the electric fields were applied to treat micewith malignant melanoma tumors. Two pairs of isolects 230 werepositioned over a corresponding pair of malignant melanomas. Only onepair was connected to the generator 210 and 200 KHz alternating electricfields (TC fields) were applied to the tumor for a period of 6 days. Onemelanoma tumor was not treated so as to permit a comparison between thetreated tumor and the non-treated tumor. After treatment for 6 days, thepigmented melanoma tumor remained clearly visible in the non-treatedside of the mouse, while, in contrast, no tumor is seen on the treatedside of the mouse. The only areas that were visible discernable on theskin were the marks that represented the points of insertion of theisolects 230. The fact that the tumor was eliminated at the treated sidewas further demonstrated by cutting and inversing the skin so that itsinside face was exposed. Such a procedure indicated that the tumor hasbeen substantially, if not completely, eliminated on the treated side ofthe mouse. The success of the treatment was also further verified byhistopathological examination.

The present inventor has thus uncovered that electric fields havingparticular properties can be used to destroy dividing cells or tumorswhen the electric fields are applied to using an electronic device. Morespecifically, these electric fields fall into a special intermediatecategory, namely bio-effective fields that have no meaningfulstimulatory and no thermal effects, and therefore overcome thedisadvantages that were associated with the application of conventionalelectric fields to a body. It will also be appreciated that the presentapparatus can further include a device for rotating the TC fieldrelative to the living tissue. For example and according to oneembodiment, the alternating electric potential applies to the tissuebeing treated is rotated relative to the tissue using conventionaldevices, such as a mechanical device that upon activation, rotatesvarious components of the present system.

Moreover and according to yet another embodiment, the TC fields areapplied to different pairs of the insulated electrodes 230 in aconsecutive manner. In other words, the generator 210 and the controlsystem thereof can be arranged so that signals are sent at periodicintervals to select pairs of insulated electrodes 230, thereby causingthe generation of the TC fields of different directions by theseinsulated electrodes 230. Because the signals are sent at select timesfrom the generator to the insulated electrodes 230, the TC fields ofchanging directions are generated consecutively by different insulatedelectrodes 230. This arrangement has a number of advantages and isprovided in view of the fact that the TC fields have maximal effect whenthey are parallel to the axis of cell division. Since the orientation ofcell division is in most cases random, only a fraction of the dividingcells are affected by any given field. Thus, using fields of two or moreorientations increases the effectiveness since it increases the chancesthat more dividing cells are affected by a given TC field.

In vitro experiments have shown that the electric field has the maximumkilling effect when the lines of force of the field are orientedgenerally parallel to the long axis of the hourglass-shaped cell duringmitosis (as shown in FIGS. 3A-3C). In one experiment, a much higherproportion of the damaged cells had their axis of division orientedalong the field: 56% of the cells oriented at or near 0° with respect tothe field were damaged, versus an average of 15% of cells damaged forcells with their long axis oriented at more than 22° with respect to thefield.

The inventor has recognized that applying the field in differentdirections sequentially will increase the overall killing power, becausethe field orientation that is most effectively in killing dividing cellswill be applied to a larger population of the dividing cells. A numberof examples for applying the field in different directions are discussedbelow.

FIGS. 27A, 27B, and 27C show a set of 6 electrodes E1-E6, and how thedirection of the field through the target tissue 1510 can be changed byapplying the AC signal from the generator 1 (shown in FIG. 1) acrossdifferent pairs of electrodes. For example, if the AC signal is appliedacross electrodes E1 and E4, the field lines F would be vertical (asshown in FIG. 27A), and if the signal is applied across electrodes E2and E5, or across electrodes E3 and E6, the field lines F would bediagonal (as shown in FIGS. 27B and 27C, respectively). Additional fielddirections can be obtained by applying the AC signal across other pairsof electrodes. For example, a roughly horizontal field could be obtainedby applying the signal across electrodes E2 and E6.

In one embodiment, the AC signal is applied between the various pairs ofelectrodes sequentially. An example of this arrangement is to apply theAC signal across electrodes E1 and E4 for one second, then apply the ACsignal across electrodes E2 and E5 for one second, and then apply the ACsignal across electrodes E3 and E6 for one second. This three-partsequence is then repeated for the desired period of treatment. Becausethe efficacy in cell-destruction is strongly dependant on the cell'sorientation, cycling the field between the different directionsincreases the chance that the field will be oriented in a direction thatfavors cell destruction at least part of the time.

Of course, the 6 electrode configuration shown in FIGS. 27A-C is justone of many possible arrangements of multiple electrodes, and many otherconfigurations of three or more electrodes could be used based on thesame principles.

Application of the field in different directions sequentially is notlimited to two dimensional embodiments, and FIG. 28 shows how thesequential application of signals across different sets of electrodescan be extended to three dimensions. A first array of electrodes A1-A9is arranged around body part 1500, and a last array of electrodes N1-N9is arranged around the body part 1500 a distance W away from the firstarray. Additional arrays of electrodes may optionally be added betweenthe first array and the last array, but these additional arrays are notillustrated for clarity (so as not to obscure the electrodes A5-A9 andB5-B8 on the back of the body part 1500).

As in the FIG. 27 embodiment, the direction of the field through thetarget tissue can be changed by applying the AC signal from thegenerator 1 (shown in FIG. 1) across different pairs of electrodes. Forexample, applying the AC signal between electrodes A2 and A7 wouldresult in a field in a front-to-back direction between those twoelectrodes, and applying the AC signal between electrodes A5 and A9would result in a roughly vertical field between those two electrodes.Similarly, applying the AC signal across electrodes A2 and N7 wouldgenerate diagonal field lines in one direction through the body part1500, and applying the AC signal across electrodes A2 and B7 wouldgenerate diagonal field lines in another direction through the bodypart.

Using a three-dimensional array of electrodes also makes it possible toenergize multiple pairs of electrodes simultaneously to induce fields inthe desired directions. For example, if suitable switching is providedso that electrodes A2 through N2 are all connected to one terminal ofthe generator, and so that electrodes A7 through N7 are all connected tothe other terminal of the generator, the resulting field would be asheet that extends in a front-to-back direction for the entire width W.After the front-to-back field is maintained for a suitable duration(e.g., one second), the switching system (not shown) is reconfigured toconnect electrodes A3 through N3 to one terminal of the generator, andelectrodes A8 through N8 to the other terminal of the generator. Thisresults in a sheet-shaped field that is rotated about the Z axis byabout 40° with respect to the initial field direction. After the fieldis maintained in this direction for a suitable duration (e.g., onesecond), the next set of electrodes is activated to rotate the field anadditional 40° to its next position. This continues until the fieldreturns to its initial position, at which point the whole process isrepeated.

Optionally, the rotating sheet-shaped field may be added (sequentiallyin time) to the diagonal fields described above, to better target cellsthat are oriented along those diagonal axes.

Because the electric field is a vector, the signals may optionally beapplied to combinations of electrodes simultaneously in order to form adesired resultant vector. For example, a field that is rotated about theX axis by 20° with respect to the initial position can be obtained byswitching electrodes A2 through N2 and A3 through N3 all to one terminalof the generator, and switching electrodes A7 through N7 and A8 throughN8 all to the other terminal of the generator. Applying the signals toother combinations of electrodes will result in fields in otherdirections, as will be appreciated by persons skilled in the relevantarts. If appropriate computer control of the voltages is implemented,the field's direction can even be swept through space in a continuous(i.e., smooth) manner, as opposed to the stepwise manner describedabove.

FIGS. 29A and 29B depict the results of in vitro experiments that showhow the killing power of the applied field against dividing cells is afunction of the field strength. In the FIG. 29A experiment, B16F1melanoma cells were subjected to a 100 kHz AC field at different fieldstrengths, for a period of 24 hours at each strength. In the FIG. 29Bexperiment, F-98 glioma cells were subjected to a 200 kHz AC field atdifferent field strengths, for a period of 24 hours at each strength. Inboth of these figures, the strength of the field (EF) is measured inVolts per cm. The magnitude of the killing effect is expressed in termsof TER, which is which is the ratio of the decrease in the growth rateof treated cells (GR_(T)) compared with the growth rate of control cells(GR_(C)).

${TER} = \frac{{GR}_{C} - {GR}_{T}}{{GR}_{C}}$

The experimental results show that the inhibitory effect of the appliedfield on proliferation increases with intensity in both the melanoma andthe glioma cells. Complete proliferation arrest (TER=1) is seen at 1.35and 2.25 V/cm in melanoma and glioma cells, respectively.

FIGS. 30A and 30B depict the results of in vitro experiments that showhow the killing power of the applied field is a function of thefrequency of the field. In the experiments, B16F1 melanoma cells (FIG.30A) and F-98 glioma cells (FIG. 30B) were subjected to fields withdifferent frequencies, for a period of 24 hours at each frequency. FIGS.30A and 30B show the change in the growth rate, normalized to the fieldintensity (TER/EF). Data are shown as mean±SE. In FIG. 30A, a windoweffect is seen with maximal inhibition at 120 kHz in melanoma cells. InFIG. 30B, two peaks are seen at 170 and 250 kHz. Thus, if only onefrequency is available during an entire course of treatment, a fieldwith a frequency of about 120 kHz would be appropriate for destroyingmelanoma cells, and a field with a frequency on the order of 200 kHzwould be appropriate for destroying glioma cells.

Not all the cells of any given type will have the exact same size.Instead, the cells will have a distribution of sizes, with some cellsbeing smaller and some cells being larger. It is believed that the bestfrequency for damaging a particular cell is related to the physicalcharacteristics (e.g., the size) of that particular cell. Thus, to bestdamage a population of cells with a distribution of sizes, it can beadvantageous to apply a distribution of different frequencies to thepopulation, where the selection of frequencies is optimized based on theexpected size distribution of the target cells. For example, the data onFIG. 30B indicates that using two frequencies of 170 kHz and 250 kHz todestroy a population of glioma cells would be more effective than usinga single frequency of 200 kHz.

Note that the optimal field strengths and frequencies discussed hereinwere obtained based on in vitro experiments, and that the correspondingparameters for in vivo applications may be obtained by performingsimilar experiments in vivo. It is possible that relevantcharacteristics of the cell itself (such as size and/or shape) orinteractions with the cell's surroundings may result in a different setof optimal frequencies and/or field strengths for in vivo applications.

When more than one frequency is used, the various frequencies may beapplied sequentially in time. For example, in the case of glioma, fieldfrequencies of 100, 150, 170, 200, 250, and 300 kHz may be appliedduring the first, second, third, fourth, fifth, and sixth minutes oftreatment, respectively. That cycle of frequencies would then repeatduring each successive six minutes of treatment. Alternatively, thefrequency of the field may be swept in a stepless manner from 100 to 300kHz.

Optionally, this frequency cycling may be combined with the directionalcycling described above. FIG. 31A is an example of such a combinationusing three directions (D1, D2, and D3) and three frequencies (F1, F2,and F3). Of course, the same scheme can be extended to any other numberof directions and/or frequencies. FIG. 31B is an example of such acombination using three directions (D1, D2, and D3), sweeping thefrequency from 100 kHz to 300 kHz. Note that the break in the time axisbetween t1 and t2 provides the needed time for the sweeping frequency torise to just under 300 kHz. The frequency sweeping (or stepping) may besynchronized with directional changes, as shown in FIG. 31A.Alternatively, the frequency sweeping (or stepping) may be asynchronouswith respect to the directional changes, as shown in FIG. 31B.

In an alternative embodiment, a signal that contains two or morefrequencies components simultaneously (e.g., 170 kHz and 250 kHz) isapplied to the electrodes to treat a populations of cells that have adistribution of sizes. The various signals will add by superposition tocreate a field that includes all of the applied frequency components.

Turning now to FIG. 14 in which an article of clothing 500 according toone exemplary embodiment is illustrated. More specifically, the articleof clothing 500 is in the form of a hat or cap or other type of clothingdesigned for placement on a head of a person. For purposes ofillustration, a head 502 is shown with the hat 500 being placed thereonand against a skin surface 504 of the head 502. An intra-cranial tumoror the like 510 is shown as being formed within the head 502 underneaththe skin surface 504 thereof. The hat 500 is therefore intended forplacement on the head 502 of a person who has a tumor 510 or the like.

Unlike the various embodiments illustrated in FIGS. 1-13 where theinsulated electrodes 230 are arranged in a more or less planararrangement since they are placed either on a skin surface or embeddedwithin the body underneath it, the insulated electrodes 230 in thisembodiment are specifically contoured and arranged for a specificapplication. The treatment of intra-cranial tumors or other lesions orthe like typically requires a treatment that is of a relatively longduration, e.g., days to weeks, and therefore, it is desirable to provideas much comfort as possible to the patient. The hat 500 is specificallydesigned to provide comfort during the lengthy treatment process whilenot jeopardizing the effectiveness of the treatment.

According to one exemplary embodiment, the hat 500 includes apredetermined number of insulated electrodes 230 that are preferablypositioned so as to produce the optimal TC fields at the location of thetumor 510. The lines of force of the TC field are generally indicated at520. As can be seen in FIG. 14, the tumor 510 is positioned within theselines of force 520. As will be described in greater detail hereinafter,the insulated electrodes 230 are positioned within the hat 500 such thata portion or surface thereof is free to contact the skin surface 504 ofthe head 502. In other words, when the patient wears the hat 500, theinsulated electrodes 230 are placed in contact with the skin surface 504of the head 502 in positions that are selected so that the TC fieldsgenerated thereby are focused at the tumor 510 while leaving surroundingareas in low density. Typically, hair on the head 502 is shaved inselected areas to permit better contact between the insulated electrodes230 and the skin surface 504; however, this is not critical.

The hat 500 preferably includes a mechanism 530 that applies a force tothe insulated electrodes 230 so that they are pressed against the skinsurface 502. For example, the mechanism 530 can be of a biasing typethat applies a biasing force to the insulated electrodes 230 to causethe insulated electrodes 230 to be directed outwardly away from the hat500. Thus, when the patient places the hat 500 on his/her head 502, theinsulated electrodes 230 are pressed against the skin surface 504 by themechanism 530. The mechanism 530 can slightly recoil to provide acomfortable fit between the insulated electrodes 230 and the head 502.In one exemplary embodiment, the mechanism 530 is a spring based devicethat is disposed within the hat 500 and has one section that is coupledto and applies a force against the insulated electrodes 230.

As with the prior embodiments, the insulated electrodes 230 are coupledto the generator 210 by means of conductors 220. The generator 210 canbe either disposed within the hat 500 itself so as to provide a compact,self-sufficient, independent system or the generator 210 can be disposedexternal to the hat 500 with the conductors 220 exiting the hat 500through openings or the like and then running to the generator 210. Whenthe generator 210 is disposed external to the hat 500, it will beappreciated that the generator 210 can be located in any number ofdifferent locations, some of which are in close proximity to the hat 500itself, while others can be further away from the hat 500. For example,the generator 210 can be disposed within a carrying bag or the like(e.g., a bag that extends around the patient's waist) which is worn bythe patient or it can be strapped to an extremity or around the torso ofthe patient. The generator 210 can also be disposed in a protective casethat is secured to or carried by another article of clothing that isworn by the patient. For example, the protective case can be insertedinto a pocket of a sweater, etc. FIG. 14 illustrates an embodiment wherethe generator 210 is incorporated directly into the hat 500.

Turning now to FIGS. 15 and 16, in one exemplary embodiment, a number ofinsulated electrodes 230 along with the mechanism 530 are preferablyformed as an independent unit, generally indicated at 540, that can beinserted into the hat 500 and electrically connected to the generator(not shown) via the conductors (not shown). By providing these membersin the form of an independent unit, the patient can easily insert and/orremove the units 540 from the hat 500 when they may need cleaning,servicing and/or replacement.

In this embodiment, the hat 500 is constructed to include select areas550 that are formed in the hat 500 to receive and hold the units 540.For example and as illustrated in FIG. 15, each area 550 is in the formof an opening (pore) that is formed within the hat 500. The unit 540 hasa body 542 and includes the mechanism 530 and one or more insulatedelectrodes 230. The mechanism 530 is arranged within the unit 540 sothat a portion thereof (e.g., one end thereof) is in contact with a faceof each insulated electrode 230 such that the mechanism 530 applies abiasing force against the face of the insulated electrode 230. Once theunit 540 is received within the opening 550, it can be securely retainedtherein using any number of conventional techniques, including the useof an adhesive material or by using mechanical means. For example, thehat 500 can include pivotable clip members that pivot between an openposition in which the opening 550 is free and a closed position in whichthe pivotable clip members engage portions (e.g., peripheral edges) ofthe insulated electrodes to retain and hold the insulated electrodes 230in place. To remove the insulated electrodes 230, the pivotable clipmembers are moved to the open position. In the embodiment illustrated inFIG. 16, the insulated electrodes 230 are retained within the openings550 by an adhesive element 560 which in one embodiment is a two sidedself-adhesive rim member that extends around the periphery of theinsulated electrode 230. In other words, a protective cover of one sideof the adhesive rim 560 is removed and it is applied around theperiphery of the exposed face of the insulated electrode 230, therebysecurely attaching the adhesive rim 560 to the hat 500 and then theother side of the adhesive rim 560 is removed for application to theskin surface 504 in desired locations for positioning and securing theinsulated electrode 230 to the head 502 with the tumor being positionedrelative thereto for optimization of the TC fields. Since one side ofthe adhesive rim 560 is in contact with and secured to the skin surface540, this is why it is desirable for the head 502 to be shaved so thatthe adhesive rim 560 can be placed flushly against the skin surface 540.

The adhesive rim 560 is designed to securely attach the unit 540 withinthe opening 550 in a manner that permits the unit 540 to be easilyremoved from the hat 500 when necessary and then replaced with anotherunit 540 or with the same unit 540. As previously mentioned, the unit540 includes the biasing mechanism 530 for pressing the insulatedelectrode 230 against the skin surface 504 when the hat 500 is worn. Theunit 540 can be constructed so that side opposite the insulatedelectrode 230 is a support surface formed of a rigid material, such asplastic, so that the biasing mechanism 530 (e.g., a spring) can becompressed therewith under the application of force and when the spring530 is in a relaxed state, the spring 530 remains in contact with thesupport surface and the applies a biasing force at its other end againstthe insulated electrode 230. The biasing mechanism 530 (e.g., spring)preferably has a contour corresponding to the skin surface 504 so thatthe insulated electrode 230 has a force applied thereto to permit theinsulated electrode 230 to have a contour complementary to the skinsurface 504, thereby permitting the two to seat flushly against oneanother. While the mechanism 530 can be a spring, there are a number ofother embodiments that can be used instead of a spring. For example, themechanism 530 can be in the form of an elastic material, such as a foamrubber, a foam plastic, or a layer containing air bubbles, etc.

The unit 540 has an electric connector 570 that can be hooked up to acorresponding electric connector, such as a conductor 220, that isdisposed within the hat 500. The conductor 220 connects at one end tothe unit 540 and at the other end is connected to the generator 210. Thegenerator 210 can be incorporated directly into the hat 500 or thegenerator 210 can be positioned separately (remotely) on the patient oron a bedside support, etc.

As previously discussed, a coupling agent, such as a conductive gel, ispreferably used to ensure that an effective conductive environment isprovided between the insulated electrode 230 and the skin surface 504.Suitable gel materials have been disclosed hereinbefore in thediscussion of earlier embodiments. The coupling agent is disposed on theinsulated electrode 230 and preferably, a uniform layer of the agent isprovided along the surface of the electrode 230. One of the reasons thatthe units 540 need replacement at periodic times is that the couplingagent needs to be replaced and/or replenished. In other words, after apredetermined time period or after a number of uses, the patient removesthe units 540 so that the coupling agent can be applied again to theelectrode 230.

FIGS. 17 and 18 illustrate another article of clothing which has theinsulated electrodes 230 incorporated as part thereof. Morespecifically, a bra or the like 700 is illustrated and includes a bodythat is formed of a traditional bra material, generally indicated at705, to provide shape, support, and comfort to the wearer. The bra 700also includes a fabric support layer 710 on one side thereof. Thesupport layer 710 is preferably formed of a suitable fabric materialthat is constructed to provide necessary and desired support to the bra700.

Similar to the other embodiments, the bra 700 includes one or moreinsulated electrodes 230 disposed within the bra material 705. The oneor more insulated electrodes are disposed along an inner surface of thebra 700 opposite the support 710 and are intended to be placed proximateto a tumor or the like that is located within one breast or in theimmediately surrounding area. As with the previous embodiment, theinsulated electrodes 230 in this embodiment are specifically constructedand configured for application to a breast or the immediate area. Thus,the insulated electrodes 230 used in this application do not have aplanar surface construction but rather have an arcuate shape that iscomplementary to the general curvature found in a typical breast.

A lining 720 is disposed across the insulated electrodes 230 so as toassist in retaining the insulated electrodes in their desired locationsalong the inner surface for placement against the breast itself. Thelining 720 can be formed of any number of thin materials that arecomfortable to wear against one's skin and in one exemplary embodiment,the lining 720 is formed of a fabric material.

The bra 700 also preferably includes a biasing mechanism 800 as in someof the earlier embodiments. The biasing mechanism 800 is disposed withinthe bra material 705 and extends from the support 710 to the insulatedelectrode 230 and applies a biasing force to the insulated electrode 230so that the electrode 230 is pressed against the breast. This ensuresthat the insulated electrode 230 remains in contact with the skinsurface as opposed to lifting away from the skin surface, therebycreating a gap that results in a less effective treatment since the gapdiminishes the efficiency of the TC fields. The biasing mechanism 800can be in the form of a spring arrangement or it can be an elasticmaterial that applies the desired biasing force to the insulatedelectrodes 230 so as to press the insulated electrodes 230 into thebreast. In the relaxed position, the biasing mechanism 800 applies aforce against the insulated electrodes 230 and when the patient placesthe bra 700 on their body, the insulated electrodes 230 are placedagainst the breast which itself applies a force that counters thebiasing force, thereby resulting in the insulated electrodes 230 beingpressed against the patient's breast. In the exemplary embodiment thatis illustrated, the biasing mechanism 800 is in the form of springs thatare disposed within the bra material 705.

A conductive gel 810 can be provided on the insulated electrode 230between the electrode and the lining 720. The conductive gel layer 810is formed of materials that have been previously described herein forperforming the functions described above.

An electric connector 820 is provided as part of the insulated electrode230 and electrically connects to the conductor 220 at one end thereof,with the other end of the conductor 220 being electrically connected tothe generator 210. In this embodiment, the conductor 220 runs within thebra material 705 to a location where an opening is formed in the bra700. The conductor 220 extends through this opening and is routed to thegenerator 210, which in this embodiment is disposed in a location remotefrom the bra 700. It will also be appreciated that the generator 210 canbe disposed within the bra 700 itself in another embodiment. Forexample, the bra 700 can have a compartment formed therein which isconfigured to receive and hold the generator 210 in place as the patientwears the bra 700. In this arrangement, the compartment can be coveredwith a releasable strap that can open and close to permit the generator210 to be inserted therein or removed therefrom. The strap can be formedof the same material that is used to construct the bra 700 or it can beformed of some other type of material. The strap can be releasablyattached to the surrounding bra body by fastening means, such as a hookand loop material, thereby permitting the patient to easily open thecompartment by separating the hook and loop elements to gain access tothe compartment for either inserting or removing the generator 210.

The generator 210 also has a connector 211 for electrical connection tothe conductor 220 and this permits the generator 210 to be electricallyconnected to the insulated electrodes 230.

As with the other embodiments, the insulated electrodes 230 are arrangedin the bra 700 to focus the electric field (TC fields) on the desiredtarget (e.g., a tumor). It will be appreciated that the location of theinsulated electrodes 230 within the bra 700 will vary depending upon thelocation of the tumor. In other words, after the tumor has been located,the physician will then devise an arrangement of insulated electrodes230 and the bra 700 is constructed in view of this arrangement so as tooptimize the effects of the TC fields on the target area (tumor). Thenumber and position of the insulated electrodes 230 will thereforedepend upon the precise location of the tumor or other target area thatis being treated. Because the location of the insulated electrodes 230on the bra 700 can vary depending upon the precise application, theexact size and shape of the insulated electrodes 230 can likewise vary.For example, if the insulated electrodes 230 are placed on the bottomsection of the bra 700 as opposed to a more central location, theinsulated electrodes 230 will have different shapes since the shape ofthe breast (as well as the bra) differs in these areas.

FIG. 19 illustrates yet another embodiment in which the insulatedelectrodes 230 are in the form of internal electrodes that areincorporated into in the form of a probe or catheter 600 that isconfigured to enter the body through a natural pathway, such as theurethra, vagina, etc. In this embodiment, the insulated electrodes 230are disposed on an outer surface of the probe 600 and along a lengththereof. The conductors 220 are electrically connected to the electrodes230 and run within the body of the probe 600 to the generator 210 whichcan be disposed within the probe body or the generator 210 can bedisposed independent of the probe 600 in a remote location, such as onthe patient or at some other location close to the patient.

Alternatively, the probe 600 can be configured to penetrate the skinsurface or other tissues to reach an internal target that lies withinthe body. For example, the probe 600 can penetrate the skin surface andthen be positioned adjacent to or proximate to a tumor that is locatedwithin the body.

In these embodiments, the probe 600 is inserted through the naturalpathway and then is positioned in a desired location so that theinsulated electrodes 230 are disposed near the target area (i.e., thetumor). The generator 210 is then activated to cause the insulatedelectrodes 230 to generate the TC fields which are applied to the tumorfor a predetermined length of time. It will be appreciated that theillustrated probe 600 is merely exemplary in nature and that the probe600 can have other shapes and configurations so long as they can performthe intended function. Preferably, the conductors (e.g., wires) leadingfrom the insulated electrodes 230 to the generator 210 are twisted orshielded so as not to generate a field along the shaft.

It will further be appreciated that the probes can contain only oneinsulated electrode while the other can be positioned on the bodysurface. This external electrode should be larger or consist of numerouselectrodes so as to result in low lines of force-current density so asnot to affect the untreated areas. In fact, the placing of electrodesshould be designed to minimize the field at potentially sensitive areas.Optionally, the external electrodes may be held against the skin surfaceby a vacuum force (e.g., suction).

FIG. 20 illustrates yet another embodiment in which a high standingcollar member 900 (or necklace type structure) can be used to treatthyroid, parathyroid, laryngeal lesions, etc. FIG. 20 illustrates thecollar member 900 in an unwrapped, substantially flat condition. In thisembodiment, the insulated electrodes 230 are incorporated into a body910 of the collar member 900 and are configured for placement against aneck area of the wearer. The insulated electrodes 230 are coupled to thegenerator 210 according to any of the manner described hereinbefore andit will be appreciated that the generator 210 can be disposed within thebody 910 or it can be disposed in a location external to the body 910.The collar body 910 can be formed of any number of materials that aretraditionally used to form collars 900 that are disposed around aperson's neck. As such, the collar 900 preferably includes a means 920for adjusting the collar 900 relative to the neck. For example,complementary fasteners (hook and loop fasteners, buttons, etc.) can bedisposed on ends of the collar 900 to permit adjustment of the collardiameter.

Thus, the construction of the present devices are particularly wellsuited for applications where the devices are incorporated into articlesof clothing to permit the patient to easily wear a traditional articleof clothing while at the same time the patient undergoes treatment. Inother words, an extra level of comfort can be provided to the patientand the effectiveness of the treatment can be increased by incorporatingsome or all of the device components into the article of clothing. Theprecise article of clothing that the components are incorporated intowill obviously vary depending upon the target area of the living tissuewhere tumor, lesion or the like exists. For example, if the target areais in the testicle area of a male patient, then an article of clothingin the form of a sock-like structure or wrap can be provided and isconfigured to be worn around the testicle area of the patient in such amanner that the insulated electrodes thereof are positioned relative tothe tumor such that the TC fields are directed at the target tissue. Theprecise nature or form of the article of clothing can vary greatly sincethe device components can be incorporated into most types of articles ofclothing and therefore, can be used to treat any number of differentareas of the patient's body where a condition may be present.

Now turning to FIGS. 21-22 in which another aspect of the present deviceis shown. In FIG. 21, a body 1000, such as any number of parts of ahuman or animal body, is illustrated. As in the previous embodiments,two or more insulated electrodes 230 are disposed in proximity to thebody 1000 for treatment of a tumor or the like (not shown) using TCfields, as has been previously described in great detail in the abovediscussion of other embodiments. The insulated electrode 230 has aconductive component and has external insulation 260 that surrounds theconductive component thereof. Each insulated electrode 230 is preferablyconnected to a generator (not shown) by the lead 220. Between eachinsulated electrode 220 and the body 1000, a conductive filler material(e.g., conductive gel member 270) is disposed. The insulated electrodes230 are spaced apart from one another and when the generator isactuated, the insulated electrodes 230 generate the TC fields that havebeen previously described in great detail. The lines of the electricfield (TC field) are generally illustrated at 1010. As shown, theelectric field lines 1010 extend between the insulated electrodes 230and through the conductive gel member 270.

Over time or as a result of some type of event, the external insulation260 of the insulated electrode 230 can begin to breakdown at any givenlocation thereof. For purpose of illustration only, FIG. 22 illustratesthat the external insulation 260 of one of the insulated electrodes 230has experienced a breakdown 1020 at a face thereof which is adjacent theconductive gel member 270. It will be appreciated that the breakdown1020 of the external insulation 260 results in the formation of a strongcurrent flow-current density at this point (i.e., at the breakdown1020). The increased current density is depicted by the increased numberof electric field lines 1010 and the relative positioning and distancebetween adjacent electric field lines 1010. One of the side effects ofthe occurrence of breakdown 1020 is that current exists at this pointwhich will generate heat and may burn the tissues/skin which have aresistance. In FIG. 22, an overheated area 1030 is illustrated and is aregion or area of the tissues/skin where an increased current densityexits due to the breakdown 1020 in the external insulation 260. Apatient can experience discomfort and pain in this area 1030 due to thestrong current that exists in the area and the increased heat andpossible burning sensation that exist in area 1030.

FIG. 23 illustrates yet another embodiment in which a furtherapplication of the insulated electrodes 230 is shown. In thisembodiment, the conductive gel member 270 that is disposed between theinsulated electrode 230 and the body 1000 includes a conductor 1100 thatis floating in that the gel material forming the member 270 completelysurrounds the conductor 1100. In one exemplary embodiment, the conductor1100 is a thin metal sheet plate that is disposed within the conductor1100. As will be appreciated, if a conductor, such as the plate 1100, isplaced in a homogeneous electric field, normal to the lines of theelectric field, the conductor 1100 practically has no effect on thefield (except that the two opposing faces of the conductor 1100 areequipotential and the corresponding equipotentials are slightlyshifted). Conversely, if the conductor 1100 is disposed parallel to theelectric field, there is a significant distortion of the electric field.The area in the immediate proximity of the conductor 1100 is notequipotential, in contrast to the situation where there is no conductor1100 present. When the conductor 1100 is disposed within the gel member270, the conductor 1100 will typically not effect the electric field (TCfield) for the reasons discussed above, namely that the conductor 1100is normal to the lines of the electric field.

If there is a breakdown of the external insulation 260 of the insulatedelectrode 230, there is a strong current flow-current density at thepoint of breakdown as previously discussed; however, the presence of theconductor 1100 causes the current to spread throughout the conductor1100 and then exit from the whole surface of the conductor 1100 so thatthe current reaches the body 1000 with a current density that is neitherhigh nor low. Thus, the current that reaches the skin will not causediscomfort to the patient even when there has been a breakdown in theinsulation 260 of the insulated electrode 230. It is important that theconductor 1100 is not grounded as this would cause it to abolish theelectric field beyond it. Thus, the conductor 1100 is “floating” withinthe gel member 270.

If the conductor 1100 is introduced into the body tissues 1000 and isnot disposed parallel to the electric field, the conductor 1100 willcause distortion of the electric field. The distortion can causespreading of the lines of force (low field density-intensity) orconcentration of the lines of field (higher density) of the electricfield, according to the particular geometries of the insert and itssurroundings, and thus, the conductor 1100 can exhibit, for example, ascreening effect. Thus, for example, if the conductor 1100 completelyencircles an organ 1101, the electric field in the organ itself will bezero since this type of arrangement is a Faraday cage. However, becauseit is impractical for a conductor to be disposed completely around anorgan, a conductive net or similar structure can be used to cover,completely or partially, the organ, thereby resulting in the electricfield in the organ itself being zero or about zero. For example, a netcan be made of a number of conductive wires that are arranged relativeto one another to form the net or a set of wires can be arranged tosubstantially encircle or otherwise cover the organ 1101. Conversely, anorgan 1103 to be treated (the target organ) is not covered with a memberhaving a Faraday cage effect but rather is disposed in the electricfield 1010 (TC fields).

FIG. 24 illustrates an embodiment where the conductor 1100 is disposedwithin the body (i.e., under the skin) and it is located near a target(e.g., a target organ). By placing the conductor 1100 near the target,high field density (of the TC fields) is realized at the target. At thesame time, another nearby organ can be protected by disposing the abovedescribed protective conductive net or the like around this nearby organso as to protect this organ from the fields. By positioning theconductor 1100 in close proximity to the target, a high field densitycondition can be provided near or at the target. In other words, theconductor 1100 permits the TC fields to be focused at a particular area(i.e., a target).

It will also be appreciated that in the embodiment of FIG. 24, the gelmembers 260 can each include a conductor as described with reference toFIG. 23. In such an arrangement, the conductor in the gel member 260protects the skin surface (tissues) from any side effects that may berealized if a breakdown in the insulation of the insulated electrode 230occurs. At the same time, the conductor 1100 creates a high fielddensity near the target.

There are a number of different ways to tailor the field density of theelectric field by constructing the electrodes differently and/or bystrategically placing the electrodes relative to one another. Forexample, in FIG. 25, a first insulated electrode 1200 and a secondinsulated electrode 1210 are provided and are disposed about a body1300. Each insulated electrode includes a conductor that is preferablysurrounded by an insulating material, thus the term “insulatedelectrode”. Between each of the first and second electrodes 1200, 1210and the body 1300, the conductive gel member 270 is provided. Electricfield lines are generally indicated at 1220 for this type ofarrangement. In this embodiment, the first insulated electrode 1200 hasdimensions that are significantly greater than the dimensions of thesecond insulated electrode 1210 (the conductive gel member for thesecond insulated electrode 1210 will likewise be smaller).

By varying the dimensions of the insulated electrodes, the pattern ofthe electric field lines 1220 is varied. More specifically, the electricfield tapers inwardly toward the second insulated electrode 1210 due tothe smaller dimensions of the second insulated electrode 1210. An areaof high field density, generally indicated at 1230, forms near theinterface between the gel member 270 associated with the secondinsulated electrode 1210 and the skin surface. The various components ofthe system are manipulated so that the tumor within the skin or on theskin is within this high field density so that the area to be treated(the target) is exposed to electric field lines of a higher fielddensity.

FIG. 26 also illustrates a tapering TC field when a conductor 1400(e.g., a conductive plate) is disposed in each of the conductive gelmembers 270. In this embodiment, the size of the gel members 270 and thesize of the conductors 1400 are the same or about the same despite thedifferences in the sizes of the insulated electrodes 1200, 1210. Theconductors 1400 again can be characterized as “floating plates” sinceeach conductor 1400 is surrounded by the material that forms the gelmember 270. As shown in FIG. 26, the placement of one conductor 1400near the insulated electrode 1210 that is smaller than the otherinsulated electrode 1200 and is also smaller than the conductor 1400itself and the other insulated electrode 1200 is disposed at a distancetherefrom, the one conductor 1400 causes a decrease in the field densityin the tissues disposed between the one conductor 1400 and the otherinsulated electrode 1200. The decrease in the field density is generallyindicated at 1410. At the same time, a very inhomogeneous taperingfield, generally indicated at 1420, changing from very low density tovery high density is formed between the one conductor 1400 and theinsulated electrode 1210. One benefit of this exemplary configuration isthat it permits the size of the insulated electrode to be reducedwithout causing an increase in the nearby field density. This can beimportant since electrodes that having very high dielectric constantinsulation can be very expensive. Some insulated electrodes, forexample, can cost $500.00 or more; and further, the price is sensitiveto the particular area of treatment. Thus, a reduction in the size ofthe insulated electrodes directly leads to a reduction in cost.

As used herein, the term “tumor” refers to a malignant tissue comprisingtransformed cells that grow uncontrollably. Tumors include leukemias,lymphomas, myelomas, plasmacytomas, and the like; and solid tumors.Examples of solid tumors that can be treated according to the inventioninclude sarcomas and carcinomas such as, but not limited to:fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, andretinoblastoma. Because each of these tumors undergoes rapid growth, anyone can be treated in accordance with the invention. The invention isparticularly advantageous for treating brain tumors, which are difficultto treat with surgery and radiation, and often inaccessible tochemotherapy or gene therapies. In addition, the present invention issuitable for use in treating skin and breast tumors because of the easeof localized treatment provided by the present invention.

In addition, the present invention can control uncontrolled growthassociated with non-malignant or pre-malignant conditions, and otherdisorders involving inappropriate cell or tissue growth by applicationof an electric field in accordance with the invention to the tissueundergoing inappropriate growth. For example, it is contemplated thatthe invention is useful for the treatment of arteriovenous (AV)malformations, particularly in intracranial sites. The invention mayalso be used to treat psoriasis, a dermatologic condition that ischaracterized by inflammation and vascular proliferation; and benignprostatic hypertrophy, a condition associated with inflammation andpossibly vascular proliferation. Treatment of other hyperproliferativedisorders is also contemplated.

Furthermore, undesirable fibroblast and endothelial cell proliferationassociated with wound healing, leading to scar and keloid formationafter surgery or injury, and restenosis after angioplasty or placementof coronary stents can be inhibited by application of an electric fieldin accordance with the present invention. The non-invasive nature ofthis invention makes it particularly desirable for these types ofconditions, particularly to prevent development of internal scars andadhesions, or to inhibit restenosis of coronary, carotid, and otherimportant arteries.

In addition to treating tumors that have already been detected, theabove-described embodiments may also be used prophylactically to preventtumors from ever reaching a detectable size in the first place. Forexample, the bra embodiment described above in connection with FIGS. 17and 18 may be worn by a woman for an 8 hour session every day for aweek, with the week-long course of treatment being repeated every fewmonths to kill any cells that have become cancerous and started toproliferate. This mode of usage is particularly appropriate for peoplewho are at high risk for a particular type of cancer (e.g., women with astrong history of breast cancer in their families, or people who havesurvived a bout of cancer and are at risk of a relapse). The course ofprophylactic treatment may be tailored based on the type of cancer beingtargeted and/or to suit the convenience of the patient. For example,undergoing a four 16 hour sessions during the week of treatment may bemore convenient for some patients than seven 8 hour session, and may beequally effective.

Example 2

Experiments were also performed on two different types ofbacteria—Pseudomonas aeruginosa strain PAO1 and Staphylococcus aureusstrain SH1000. All strains were grown in LB media (1.0% Bacto tryptone,0.5% Yeast extract, 1% NaCl). Broth cultures of freshly plated bacteriastrains were grown in 3 ml of liquid medium at 37° C. for 15 hours in anorbital shaker (220 RPM) and diluted in fresh LB broth to apredetermined absorbance at 595 nm which yielded the desired CFU per ml.

FIG. 32A depicts the construction of the electrodes 1610 used in theexperiment. Each electrode is 15 mm long and 5 mm high. It include anelectrical conductor 1611 with its outer face coated with a thin layerof lead magnesium niobate-lead titanate (PMN-PT) ceramic insulation1612, which has a high dielectric constant (∈>5000) such that theircapacitance was about 10 nF each. The rear of the conductor 1611 wasinsulated using a 5 mm layer 1614 of 353ND medical grade epoxy (EpoxyTechnology, Billerica, Mass., USA) and a wire 1613 is connected to theconductor 1611. Of course, it may be appropriate to vary the dimensionsof the electrodes depending on the intended application.

FIG. 32B depicts a test chamber that includes four electrodes 1610,arranged in pairs and positioned in a 50 mm Petri dish 1626. Theelectrodes were held in place by a polycarbonate holder 1624. Electricfields were generated in the test chamber by applying an AC voltageacross one pair of opposing electrodes, then applying an AC voltageacross the other pair of opposing electrodes, in an alternating sequenceto produce electric fields in the medium that are oriented at 90° withrespect to each other. The electrodes were placed 23 mm apart. Theelectrodes were completely insulated from the medium in the Petri dishby the ceramic insulation 1614 on the face of the electrode 1610, so thefield is capacitively coupled through the layer 1614 into the targetregion.

FIG. 32C depicts a setup that was used to induce fields in the testchamber 1620. The output of a sinewave generator 1632 (Model 662, OR-X,Israel) is routed to an RF amplifier 1634 (75A250, AR worldwide,Souderton, Pa., USA), and the output of the RF amplifier 1634 is routedto a field direction switching relay 1636 that either imposes theamplified sine wave between the upper and lower electrodes or betweenthe right and left electrodes. The switching relay is configured toswitch back and forth between those two states periodically, therebyswitching the direction of the field at the desired interval. The entirefield generating system was placed inside a Faraday cage 1644 in orderto meet the guidelines for limiting exposure to time-varying electric,magnetic, and electromagnetic fields of the International Non-IonizingRadiation Committee (INIRC).

Temperature was measured continuously using insulated T typethermocouple (Omega, Stamford, Conn.) with its tip positioned at thecenter of the chamber 1620. The thermocouples were connected to a TC-08Thermocouple Data Logger (Pico Technologies, UK) the output of which wasconnected to computer 1630. When high field frequencies (30-50 MHz) wereused, the fields interfered with the temperatures measurements. So tomeasure the temperature, the field was temporarily turned off for twoseconds during each temperature measurement.

As electric fields are associated with heat production, the chambertemperature was held at the desired value by computer feedback controlof the amplitude of the waveform at the input of the power amplifier.The electric field intensities in the culture medium were measured usinga shielded coaxial probe having two exposed tips fixed at a distance of1 cm. The probe was connected, through a coaxial cable, to a 190Bfloating scope meter (Fluke, The Netherlands). Field intensities weremeasured at the end of each treatment by dipping the probe in theculture media, such that the two measuring points were in parallel withthe lines of the electric field. Field intensities are expressed aspeak-to-peak voltage per centimeter distance (V/cm).

Overnight bacterial cultures were diluted in fresh LB broth to an ODthat corresponds to bacterial counts of 1×10⁷ Colony Forming Units(CFU)/ml. Petri plates containing the electric fields chamber 1620 werefilled with 7 ml of the diluted cultures, and placed inside a pre-cooledincubator (FOC 2251, Velp Scientifica). Fields were applied for 2 hoursfor S. aureus and 2.5 hours for P. aeruginosa with the field directionalternating every 300 ms (i.e., 300 ms in one direction followed by 300ms in the other direction). Preliminary experiments indicated that thesedurations were sufficient to allow for approximately one order ofmagnitude growth of the control (unexposed to electric fields) group.

Temperature within the chamber 1620 was controlled by modifying thefield strength within a predetermined range since the field causesheating. The electric fields chamber temperature reached 37±0.2° C.within the first 5 minutes of the experiment in both the treated groupand in the control group. (The control bacteria groups were not exposedto the electric fields, but were temperature-controlled to match thetemperature in the test groups.)

At the end of treatment, the cultures were stirred several times by upand down pipetting. Four aliquots of 250 μl were dispensed into a 96MicroWells plate (Nunclon™A, Nunc, Denmark) and the OD was determinedspectrophotometrically with a microplate reader (Infinite 200, Tecan,Austria) at 750 nm. The optical densities (ODs) of the blanks, whichconsisted of uninoculated LB, were subtracted from the ODs of theinoculated plates. The percentage of growth for each well was calculatedby dividing the OD of the wells by that of the control: (od_(750nm) oftreated wells/_(OD750nm) of the control well)×100.

The effect of the electric fields' frequency was tested by applyingfields between 100 kHz and 50 MHz. The results, depicted in FIGS. 33Aand 33B, show that 2-4V/cm electric fields inhibit the growth of the S.aureus (after 2 hours treatment) and P. aeruginosa (after 2.5 hourstreatment), respectively. The calculated effect, expressed in % (usingthe scale on the left side of the graphs), is based on OD measurements.Averages of at least two independent experiments±standard errors arepresented. The corresponding average field intensities±standard errorsare indicated by the solid line (using the scale on the right side ofthe graphs).

The results show that the growth inhibition is frequency dependent,having a maximum growth inhibition at 10 MHz fields for both S. aureusand P. aeruginosa. Note that as the electric fields generating systemwas designed to maintain a constant temperature in the chamber byadjustment of the field intensity, and therefore the fields' intensitiesvary between the different frequency tests. The field intensityvariations in the tests was limited to a range of ±1 V/cm. The resultspresented are means±standard errors (SE) of at least 2 independentexperiments, each consisting of 6-8 plates. Higher field frequencieswere not tested due to equipment limitations.

The effect of the electric fields' intensity was tested by applying 10MHz fields to S. aureus at different intensities for 6 hours. Therelative growth, based on CFU counts, is expressed as a percentage ofthe heat control±standard errors. The initial S. aureus concentration inthis set of experiments was 0.5-1×10⁵ CFU/ml. As seen in FIG. 34, thegrowth inhibition is field intensity dependent reaches a plateau of justabove an 80% inhibition, at field intensities of about 2-2.5 V/cm. Notethat although this intensity worked best for treating S. aureus at 10MHz, field strengths between 0.5 and 10 V/cm may be used.

The effect of the switching rate of the electric fields between the twoperpendicular directions was tested by applying 10 MHz, 3.5 V/cm fieldsand varying the switching rate (i.e., the time the field was applied ineach direction). The dependency of the fields' inhibitory effect isillustrated in FIGS. 35A and 35B for S. aureus and P. aeruginosa,respectively (after treatment for 2 hours for S. aureus or 2.5 hours forP. aeruginosa). The results indicate that for S. aureus, durations of100 mSec, 1 Sec, and 30 Sec resulted in a significantly higherinhibition than the other durations tested. In the case of P. aeruginosathe maximal inhibition was observed when the duration of each fielddirection was 30 Sec.

The combined effect of the electric fields and antibiotics was alsotested. Chloramphenicol was obtained as powder and dissolved in EtOH(99%, Frutarom). All the stock solutions were filter sterilized and heldat −20° C. until use. Serial twofold dilutions of each antibiotic agentwere prepared following NCCLS guidelines. The MIC of an antibiotic wasdefined as the lowest concentration that completely inhibited growth ofthe organism, as determined by the unaided eye. These results were inagreement with over 95% inhibition compared with that of drug-freewells, as determined using the microplate reader at 750 nm. The MIC ofelectric fields was defined as the lowest intensity that inhibits growthby 80% or more compared with control, as determined using the microplatereader.

Drug interactions with the electric fields were assessed according tothe checkerboard method, with the following modifications: S. aureusinocula were diluted in LB medium containing the antibiotic to finalconcentration of 0.5 to 1.0×10⁵ CFU/ml. The final concentrations of thechloramphenicol ranged from 0.125 to 16 ng/ml. Petri dishes containingthe electric fields chamber were filled with 7 ml of the dilutedcultures, and placed inside a pre cooled incubator. Fields were appliedfor 6 hours with the field direction alternating every 300 ms and thefield intensities were varied by changing the incubator ambienttemperature. Thus, lower ambient temperatures allowed for higher fieldintensities while maintaining the proper culture temperature of 37° C.Control plates containing the same electric fields chambers were placedin a pre-warmed incubator set at 37° C. At the end of the treatment, thecultures were stirred by pipetting the plate content up and down.Quadruplicates of 250 μl each were transferred to a 96 MicroWells plate(Nunc) and the OD was determined using Tecan microplate reader. Cultureswere subjected to serial 10-fold dilution (up to 1/10,000) by adding 20n1 of sample to 180 μl of saline solution (0.85% NaCl), from which 80 μlaliquots were plated on LB agar plates (1.5% agar, 1.0% Bacto tryptone,1% NaCl, 0.5% Yeast extract). CFU counts were performed after overnightincubation at 37° C. The results were grouped and the effect wascalculated by dividing the OD or CFU of the experiments plates withthose of the control plates.

To evaluate the effect of the combinations, the fractional inhibitoryconcentration (FIC) was calculated for the electric fields and for eachantibiotic. The following formulas were used to calculate the FIC index:

FIC of electric fields=MIC of electric fields in combination/MIC ofelectric fields alone,

FIC of drug B=MIC of drug B in combination/MIC of drug B alone,

and

FIC index=FIC of electric fields+FIC of drug B.

Synergy was defined as an FIC index of ≦0.5. Indifference was defined asan FIC index of >0.5 but of ≦4. Antagonism was defined as an FIC indexof >4.

The MIC of chloramphenicol against S. aureus was found to be 4 μg/ml,similar to the concentrations reported in the literature. The combinedeffect of electric fields and chloramphenicol on the growth of S. aureusis given in Table 1. The results demonstrate that there is an additiveeffect between electric fields and chloramphenicol. As seen, in thepresence of 4 V/cm 10 MHz electric fields applied for 6 hours, with thefield direction alternating every 300 ms, much lower concentrations ofchloramphenicol (1 μg/ml) are sufficient to produce complete inhibitionof the growth of S. aureus. The FIC index was found to be 0.625,indicating that there is an additive effect for the combined exposure toelectric fields and chloramphenicol. Note that these calculations arebased on OD measurements.

TABLE 1 0 V/cm 0.5-2.0 V/cm 2.0-2.5 V/cm 3.5-4.5 V/cm   0 μg/ml 100% 62%32% 19% 0.25 μg/ml  66% 39% 51% 13%  0.5 μg/ml  45% 16% 20% 21%   1μg/ml  30% 26%  6%  2%   4 μg/ml  7% 13%  2%  0%

Note that while the examples above used an antibiotic, other therapeuticagents may be substituted for antibiotics in appropriate situations.

Tests were also performed to determine whether repeated exposure of P.aeruginosa to electric fields could select for bacteria that areresistant to the fields. For this test, overnight bacterial cultureswere diluted in fresh LB broth to an OD that corresponds to bacterialcounts of 1×10⁶ CFU/ml. Bacteria were exposed to 10 MHz electric fieldsof approximately 5 V/cm for 6 hours as described above, with the fielddirection alternating every 300 ms. Control bacteria groups, placed ininactivated electric fields chambers, were positioned in a pre-warmedincubator. The electric fields' effect was determined based on the ODmeasurements. After the initial electric fields inhibition experiment,the fields' effect was determined anew daily, for four passages asfollows: samples from the plates treated with electric fields werepooled and used again for electric fields effect determination in thesubsequent generation. In parallel, the fields' effect evolution duringthese subcultures was compared concomitantly with each new generation,using bacteria harvested from control wells (wells cultured in thepre-warmed incubator). The relative effect was calculated for eachexperiment from the ratio of inhibition obtained for a given subcultureto that obtained for first-time exposure.

The results of this repeated exposure test of P. aeruginosa to electricfields are presented in FIG. 36. As demonstrated, four passages ofexposure to electric fields did not result in development of resistance,and the inhibition percentage remained around 70% in each iteration.

Numerical calculations, based on finite element mesh, were used in orderto calculate the electric field distribution inside dividing P.aeruginosa and S. aureus, and the following geometries and parameterswere used for the calculations: P. aeruginosa was assumed to be anellipse with a large radius of 2.0 μm and a small radius of 0.6 μmhaving two membranes (external and internal) of 8 nm thickness. The twomembranes were assumed to be separated by a periplasmic space of 50 nm.The dividing bacterium furrow diameter was assumed to be 0.2 μm and theapplied external field was 20 V/cm. Since no published data on theelectric properties of P. aeruginosa was found, the following data forE. coli was substituted in the calculations: inner membraneconductivity—1 μS/m, outer membrane conductivity—3 mS/m, mediumconductivity—0.5 S/m, cytoplasm conductivity—0.5 S/m and theconductivity of the periplasmic space—50 mS/m.

S. aureus was assumed to be a sphere with a radius of 0.6 μm and amembrane thickness of 8 nm. The bacterial cell wall thickness wasassumed to be 20 nm, and the dividing bacterium furrow diameter wasassumed to be 0.2 μm. The applied external field was 20 V/cm. In thesimulation the membrane conductivity was assumed to be 1 μS/m, and thecell wall conductivity 10 mS/m. The conductivity of the medium was 0.5S/m and the conductivity of the cytoplasm was assumed to be 0.8 S/m.

The electric field distribution in and around P. aeruginosa and S.aureus was calculated using finite element mesh method, and the resultsare depicted in FIGS. 37 and 38. In the simulation (FIG. 37) it is seenthat the inside dividing rod-like bacteria, close to the furrow, theelectric field is strongest and is non uniform. This non uniformitygenerates dielectrophoresis forces. When the field intensity is 20 V/cm,the magnitude of the Force acting on a dipole of 3000 debyes insidedividing bacteria as a function of field frequency is depicted in FIG.38A for P. aeruginosa, which peaks at about 2 MHz, and depicted in FIG.38B for S. aureus, which peaks at about 7 MHz.

In vivo tests were also performed to test the ability of electric fieldsto inhibit the growth of bacterial pathogen in vivo. 2×10⁸ S. aureusbacteria were injected S.C. into the dorsum of 8 weeks old female ICRmice. Mice in which abscess was developed in the site of injectionwithin 24 hours were anesthetized and 4 electrodes were placed on theirback. The electrodes were similar to those described above but alsocontained a thermistor positioned inside the electrodes near theelectrode surface. The electrodes were arranged in two electrode pairspositioned perpendicular to each other so as to generate electric fieldsin two different directions, spaced 90° apart, and the distance betweeneach electrode within any given pair was 2 cm. Note, however, thatalternative electrode configurations may be used, e.g., as discussedabove in connection with FIGS. 27 and 28.

Mice in the control group were identical to those in the electric fieldsgroup, but carrying heating electrodes. Mice were held in an IVC system(Techniplast, Italy) whose cages were modified in order to allow forelectric fields application inside the cage. 10 MHz electric fields wereapplied for 48 hours at a nominal field strength of about 5 V/cm, withthe field direction alternating every 300 ms, and the temperature at theelectrode surface was monitored continuously. The electrodes temperaturewas held at the desired value by computer feedback control of theamplitude of the waveform at the input of the power amplifier. Shamcontrol electrodes were heated to the same temperature as the electricfields electrodes. No adverse side effects were observed. After 48 hoursthe mice were anesthetized, sacrificed, and the electrodes were removed.A 2×2 cm square of the skin surrounding the abscess was harvested,weighed and homogenized. The homogenate was serially diluted and platedin triplicates for CFU determination. The initial S. aureusconcentration in these experiments was 2×10⁷ CFU/ml. The results of thisexperiment are depicted in FIG. 39, which indicate that the electricfields can inhibit bacterial growth in mature abscesses.

Depending on the location of the target region within the body, theelectrodes may be either placed on the patient's body or implanted inthe patient's body. For example, in a patient with an infected cyst, theelectrodes could be implanted near the cyst. Note that theMegahertz-range frequencies that were found to be effective againstbacteria have virtually no impact on eukaryotic cells, so specificity isexcellent, and adverse side effects are not a major concern. Optionally,different frequencies may be applied to the target region, eithersimultaneously or sequentially, to target one or more types of bacteriathat may be present, as discussed above in connection with the otherembodiments. The method may also be used in vitro, e.g., to combatbacteria in food, on media, cell cultures, etc.

Example 3

In vivo experiments were also performed on Pseudomonas aeruginosa strainPAo1 in lungs of infected mice. For this example, bacteria were grown inLB media (1.0% Bacto tryptone, 0.5% Yeast extract, 1.0% NaCl (Frutarom,Haifa, Israel). Broth cultures of freshly plated bacteria were grown in250 ml of liquid medium at 37° C. in an orbital shaker at 220 RPM (NewBrunswick Scientific, N.J., USA) up to logarithmic phase, centrifugedand resuspended in saline containing 15% glycerol. Stocks were dividedinto aliquots of 50 μl each containing 2.17×10⁷ Colony Forming Units(CFU)/ml and kept at −70° C. until use.

Experiments were conducted on 7- to 8-week-old (body weight 26-35 g) ICRfemale mice obtained from Harlan laboratories (Israel). Mice were housedunder standard conditions of light and temperature, and were fedstandard laboratory chow and water ad libitum. Electric fields weregenerated by 2 pairs of parallel metal electrodes, 15 mm-long; 3mm-high. The front of the electrodes was completely insulated from themedium by a ceramic [(lead magnesium niobate-lead titanate (PMN-PT)(EDO, New York, N.Y.)] having a very high dielectric constant (∈>5000)such that their capacitance was about 10 nF each. The back of theelectrodes was insulated by a 2.5 mm thick layer of 353ND medical gradeepoxy (Epoxy Technology, Billerica, Mass., USA). Each electrodecontained a thermistor placed within the epoxy directly on top of theceramic in order to allow for constant monitoring of the electrodes'temperature.

The four insulated electrodes were attached to the skin using hydrogelafter mouse depilation. The electrodes were wrapped with leucoplast andelectrodes wires were connected to the Electric fields generating systemor to the sham control system. The four electrodes were functionallydivided into two pairs, identified by the red or blue wires. Theelectrode pairs were placed so as to create two perpendicular fielddirections at the center of the animal body.

In order to allow for electric fields generation in mice, whilemaintaining the strict isolation required when working with animalinfected with pathogens, the mice were held in an IndividuallyVentilated Cage system (IVC) (TouchSLIM™, Techniplast, Italy) that wasmodified as follows: Each cage (GR900, Techniplast, Italy) was split bya polycarbonate partition into two units, each housing one mouse. Eachmouse was treated with a separate field generator consisting of an RFamplifier (75A250, AR worldwide, Souderton, Pa., USA) activated by asine-wave generator (Model 662, OR-X, Israel). Six hermetically sealed,shielded electric connection sockets (SM3416 BNC, S.M. electronics, TX,USA) were positioned at the front panel of the cage. Each socket allowedfor the connection (by means of coax cables) of a connection box placedinside the cage to an exterior device; an RF amplifier or a temperaturemeasuring system. The connection box served for transferring theelectric currents to the mice electrodes, and the mice temperaturereadout to the computer.

The entire IVC system, together with the electric fields generatingsystem, were placed inside a Faraday cage in order to meet theguidelines for limiting personnel exposure to time-varying electric,magnetic, and electromagnetic (International Non-Ionizing RadiationCommittee—INIRC). To avoid field interference with temperaturemeasurements, temperatures were measured periodically while the fieldwas briefly turned off.

As electric fields are associated with heat production, the electrodessurface temperature was kept constant at the desired level by computerfeedback control of the field amplitude. The current source output wasswitched, every 300 ms, between the two electrodes pairs. This intervalwas found previously to be effective against P. aeruginosa whilepreventing the formation of temperature gradients that could affectbacterial growth rate (Giladi et al., 2008). The lung temperature wasmeasured periodically by inserting a thermocouple connected to atemperature data logger (TC08 USB) (Pico technology, Cambridgeshire, UK)into the mouse trachea.

Two control groups were set in each experiment: sham heat control, andnon treated control (referred to as “Control”). Control electrodes ofidentical size and shape to the electric fields electrodes, were placedon mice of the two control groups. The Sham heat electrodes producedequal temperature changes to those produced by the field electrodes bymeans of a heating resistor incorporated within them, and the sham heatelectrodes were placed in the same positions as the real electrodes.Except for the differences in the electrodes, the control mice and thesham heat treated mice were held in conditions identical to those of theelectric fields treated mice.

The electric field intensity was measured using a shielded coaxial cableand probe having two exposed tips positioned at a distance of 1 cm fromeach other. The probe was connected to a floating input oscilloscope(190B Fluke, The Netherlands). Field intensities within lungs weremeasured by performing a 5 mm long incision in the thoracic skin of ananesthetized mouse and inserting the measurement probe at an intercostalspace to a depth of 5 mm into the thorax. The probe was oriented suchthat the line connecting the two measuring points was parallel to thelines of the electric field. Field intensities are expressed aspeak-to-peak volts per centimeter distance (V/cm).

Mice were rendered neutropenic (neutrophil counts, <100/mm³), asdescribed in Andes and Craig (1998), by two intraperitoneal injectionsof cyclophosphamide; four days before infection (150 mg/kg of bodyweight) and an additional injection one day before infection (100 mg/kgof body weight). The pulmonary infection model of Takeda et al., (2006)was used with minor modifications: Anesthetized ICR mice were challengedintra-nasally with 20 μL of saline containing various concentrations(10⁴-10⁷ CFU/ml) of P. aeruginosa strain PAo1. On the day of infection,immediately before use, frozen bacterial cultures were thawed anddiluted in saline to the desired concentration. Anesthetized mice werechallenged intra-nasally with the bacterial suspension. Mice weresacrificed at 0, 3, 6, 9, 24, and 48 hours after infection in order todetermine CFU counts in their lungs. After skin disinfection with 70%alcohol, lungs from sacrificed mice were removed aseptically,photographed, weighted and their CFU content was determined.

Application of electric fields to the mouse torso was carried out asfollows: 4 days before infection, the torso of anesthetized mice wasshaved with electrical clippers and depilated using cold wax. Electricfields treatment was applied through four insulated electrodes placedaround the mouse torso so as to generate fields of two perpendiculardirections. Mice were placed inside the IVC cages and the electrodeswere connected to the connection box. Application of electric fieldsbegan 3 hours after infection and was maintained for 48 h with two briefstops daily for monitoring the physical condition of the mice. After 48h of treatment, the mice were sacrificed and bacterial numbers in lungwere determined.

The combined effect of electric fields and antibiotics was tested usingCeftazidime, an agent recommended by the infectious disease society ofAmerica for the treatment of hospital acquired pneumonia.

Experiments were conducted as described above for electric fieldstreatment of P. aeruginosa pulmonary infection with the followingmodifications: mice were challenged intra-nasally with a predeterminedbacterial titer. Ceftazidime (5 mg/kg body weight) was administeredintra-peritoneally twice daily for 48 hours starting 3 h afterinfection. Antibiotic administration was stopped at least 12 h beforeexperiment termination.

Cold sterile saline was added to each lung sample tube, saline:tissueweight ratio being 10:1. Tubes were placed in an ice bath and thesuspensions were homogenized using Omni tissue homogenizer (Omni, Ga.,USA) with Omni disaggregation tips. Appropriate suspension dilutionswere plated on LB agar plates and kept at 37° C. for 24 h. Bacterialcounts were expressed as number of P. aeruginosa CFU per lung.

Bacterial growth in the lungs of each mouse was expressed using thefollowing formula: log(CFU_(T48)/CFU_(T0)), where CFU_(T48) is the CFUcounts in the mouse lungs at the end of treatment and CFU_(T0) is theinitial number of bacteria used for inoculation in the specificexperiment. The results of all mice that received the same treatmentwere pooled and the average bacterial growth was calculated for eachgroup. T test with two tailed distribution and equal variance was usedto compare the mean values.

Lungs were first inflated with formaldehyde to their normal volume andthen underwent fixation. The specimens were processed and embedded inparaffin wax. Histological sections of 5 μm were cut, stained withhaematoxylin-eosin (HE) and examined using light microscopy by anindependent expert who was blinded to the source of the sections. Asemi-quantitative grading scheme was used to evaluate the extent of thelesions in the sections as follows: minimal (grade 1) lesions involvingless than 10% of the lung section; mild (grade 2), 11%-40%; moderate(grade 3), 41%-80%; marked (grade 4), 81%-100%.

The results of the experiments are described below. Field intensitiesand temperature measurements were done during electric fields treatmentand sham heat application. The average electric fields intensities inthe lungs were 12±4 V/cm. Under these conditions lung temperature wasidentical to, or up to 1° C. lower than the electrode surfacetemperatures which were set to 37° C. for both the electric fields andthe sham heat control.

Pulmonary infection was induced by intranasal administration ofdifferent amounts of P. aeruginosa strain PAO1 to neutropenic ICR mice.Bacteria concentrations that were found to induce a severe pulmonaryinfection which persisted for at least 48 h were around 5×10⁴ CFU/mouse.Higher concentrations (5×10⁵ CFU/mouse) resulted in a high mortalityrate (100% within the first 24 h) while lower concentrations (5×10³CFU/mouse) resulted in a complete recovery.

Table 2 shows the electric fields' effect on mice with P. aeruginosalung infection, used alone and combined with ceftazidime. The log CFU atboth the start of treatment (T0) and 48 hours later (T48) is listed intable 2. Table 3 shows the electric fields' effect on mice with P.aeruginosa lung infection, used alone and combined with ceftazidime. Thegrowth listed in table 3 is relative to the inoculums after 48 hours oftreatment. Table 4 shows the electric fields' effect on mice with P.aeruginosa lung infection, used alone and combined with ceftazidime. Thelung weights listed in table 4 are the weights at the end of the 48 hourtreatment.

TABLE 2 Number of bacteria (CFU) in the lungs of infected mice withoutceftazidime with ceftazidime Log CFU Log CFU P values Log CFU Log CFU Pvalues (T0) ± (T48) ± compared # of (T0) ± (T48) ± compared # of STDEVSTDEV to control mice STDEV STDEV to control mice fields 3.6 ± 0.2 4.1 ±1.5 <0.001 17 4.6 ± 0.2 5.0 ± 1.7 0.005 36 sham heat 3.6 ± 0.2 5.0 ± 2.50.009 20 4.6 ± 0.2 6.0 ± 2.3 0.398 27 control 3.6 ± 0.2 7.5 ± 2.7 24 4.6± 0.2 6.5 ± 2.5 40

TABLE 3 P. aeruginosa growth in mice lungs without ceftazidime withceftazidime log CFU/lung P values log CFU/lung P values (T48/T0)compared (T48/T0) compared (±STDEV) to Control (±STDEV) to Controlfields 0.5 ± 1.6 <0.001 0.4 ± 1.7 0.004 sham heat 1.6 ± 2.5 <0.01 1.3 ±2.2 0.4 control 3.8 ± 2.8 1.9 ± 2.5

TABLE 4 Average lungs weight in mice with pulmonary infection aftertreatment without ceftazidime with ceftazidime P values P values weightin mg compared weight in mg compared (±STDEV) to Control (±STDEV) toControl fields 257 ± 32 <0.001 244 ± 31 <0.001 sham heat 290 ± 49 <0.001275 ± 60 0.047 control 377 ± 97 310 ± 77

48 hours electric fields treatment of mice suffering from pulmonaryinfection resulted in 3.4 log reduction in the CFU content of thetreated mice as compared to the untreated control mice (see Table 2).The sham heated control treatment resulted in 2.5 log reduction ascompared to the control mice (see Table 2). The average bacterial growth[log(CFU_(T48)/CFU_(T0))] of both electric fields treated mice and thesham heat treated mice was significantly lower than in the control mice(see Table 3). A significant reduction was also observed in the weightof lungs of electric fields treated mice as compared to both the shamheated control mice (P=0.02) and to the untreated control mice (P<0.001)(see Table 4). Finally, histopathology examination of the lung samplesof the mice that were treated with electric fields were rated as normalto grade 1 with focal acute inflammation. In contrast, lungs of controlmice or the sham heat group were rated as grade 2 to 3 associated withacute necrotizing pneumonia and the presence of numerous bacterialcolonies.

Experiments were also performed to test the effects of combinedtreatment of pulmonary infection caused by P. aeruginosa by electricfields combined with ceftazidime. The inoculums that were found toinduce a persistent pulmonary infection in the presence of ceftazidime(10 mg/kg BW/day) alone were around 5×10⁵ CFU per mouse. The resultspresented in Table 2 demonstrate that 48 h combined treatments ofelectric fields and ceftazidime resulted in a 1.5 log reduction in thelung CFU content compared to the antibiotic-only treated control. Thecombined treatment of sham heat and ceftazidime did not result in asignificant count reduction compared to the control (see Table 2).Bacterial growth [log(CFU_(T48)/CFU_(T0))] in the lungs of mice treatedwith electric fields and ceftazidime was significantly lower than in thecontrol mice (P=0.004) (see Table 3). The average lung weight wassignificantly lower in both the ceftazidime/electric fields combinationtreatment group (P<0.001) and in the ceftazidime/sham heat combinationtreatment group (P=0.047) as compared to the control group (see Table4).

The results obtained demonstrate that electric fields, previously shownto be an effective in-vitro tool for combating bacterial pathogens(Giladi et al., 2008), are also effective in the in-vivo arena.Specifically, it was demonstrated that electric fields, applied by meansof external insulated electrodes, have a significant inhibitory effecton the growth of a bacterial pathogen in-vivo. The inhibitory effect wasobserved when electric fields were applied either as a sole treatment oras an adjuvant to antibiotic therapy.

One striking outcome of the electric fields application was the factthat the number of bacteria in the lungs of electric fields treated micewas increased only by a fraction of an order of magnitude compared tothe number in the inoculums used for infection, while at the same timethe number of bacteria in the control group grew by 3.8 orders ofmagnitude. This observation could be the consequence of either anelectric fields inhibitory effect or a bactericidal effect.Histopathology analysis demonstrated that in the electric fields treatedmice, the lung morphology was normal with no evidence of a formermassive infection. Thus it is reasonable to assume that the electricfields treatment led to cell proliferation arrest, rather than to theelimination of the bacteria after thriving in the lungs. Supportiveevidence for this conclusion can be found in the results of theexperiment in which combined electric fields/ceftazidime treatment wastested. Ceftazidime is a third-generation cephalosporin antibiotic witha broad bactericidal activity against Gram positive and Gram negativebacteria. Like other beta-lactam antibiotics, ceftazidime is mosteffective against dividing bacteria. Indeed, ceftazidime treatment of 10mg/kg BW/day, alone, resulted in a growth reduction of close to 2 logs,compared to the non treated lungs (1.9 vs. 3.8 respectively, see Table3). Yet, the combined treatment of electric fields and ceftazidime didnot lead to a reduction in the bacterial growth compared to the electricfields treatment alone (0.4 vs. 0.5, respectively, see Table 3), nor didit reduce CFU counts in the lungs compared to the inoculums suggestingthat bacterial growth was arrested.

Previously we demonstrated that application of electric fields in vitroled only to a 20% reduction in the number of treated bacteria comparedto the control, which is considerably less than the inhibition thatoccurred in the in vivo study discussed herein. One possible explanationfor the enhanced efficacy in the in vivo experiments could be derivedfrom the fact that the electric Fields inhibitory effect is positivelycorrelated with field intensity (Giladi et al., 2008). While theelectric fields intensity in the in vitro experiments was limited to 4V/cm, in order to avoid over heating of the culture, the electric fieldsintensity in the in vivo experiments was significantly higher (i.e.,about 12 V/cm). The application of such electric fields with higherintensities was made possible in view of the active heat removal thatoccurs in the mouse's body.

Experimental data obtained for both microorganisms and proliferatingliving cells, such as cancer cells, indicates that higher fieldintensities result in higher efficacy. However, there is a practicallimit on field intensity due to the heat generated under the electrodesand within the treated animal, and the relevant anatomy's ability tocarry that heat away.

For example, in the case of a human brain, the blood can carry away theheat that is generated in the tissue, and the total blood flow is about2000 cc/min. Assuming that we want to keep the temperature of the brainbelow 39° C., which is 2° above the normal body temperature, this bloodflow should be able to carry away 4000 calories/min, which correspondsto about 280 W. We should therefore be able to apply 278 W safely to thebrain. Since the side to side electric resistance of the human brain isabout 100 Ohms, and power equals voltage squared divided by resistance,we should be able to use a voltage of 167 V safely. This voltage dropsover a distance of about 10 cm, in which case the field strength will beabout 17 V/cm. Thus, for the brain, it would be reasonable to operate atfield strengths between 5 and 20 V/cm, and more preferably at fieldstrengths between 10 and 17 V/cm.

In the case of resting skin, the blood flow is sufficient to handlefields of 5V/cm, and in the case of hyperemic heated skin the blood flowis sufficient to handle fields of 50 V/cm or even 80 V/cm. It istherefore expected that practical useful fields will range from 2 V/cmto 100V/cm, depending on the treated organ and its state. Note that theabove calculations are for RMS Voltage, and not peak to peak voltage.

Another possible factor for the enhanced efficacy of the electric fieldstreatment in the in vivo arena could be related to the fact that thesham heat treatment also led to an inhibitory effect on bacterialgrowth. Though the sham heat control effect was smaller than theelectric fields effect, it was nevertheless significant. Bacterialgrowth inhibition due to elevated temperatures may result from either adirect effect of the heat on bacterial growth or from local and systemic“host” reactions and responses to the heating. The former hypothesis wastested in vitro by comparing the growth rate of P. aeruginosa at varioustemperatures within the range of normal to febrile temperatures. Similarto the reports in the literature (Magnusson et al., 1995), we found thatthe growth rate of P. aeruginosa is positively correlated with increasein the temperature in the range of 34-38° C. Thus we can rule out thepossibility of a direct effect of heat on bacterial growth. Thealternative notion, i.e. that local and systemic host reactions toelevated temperatures negatively affect bacterial growth is not new. Infact, heat and fever were used as a tool against microbial pathogens forover a century (reviewed by Atkins, 1982 and Hasday et al., 2000). Thereare numerous reports regarding the effect of elevated host temperatureson the growth of bacterial pathogens demonstrating for example that:fever induced after Malaria inoculation can serve as a treatment againstTreponema pallidum (Wagner-Jauregg, 1919), febrile core temperature isessential for optimal host defense against Klebsialla pneumoniaperitonitis in mice (Jiang et al., 2000), fever in elderly patients withcommunity-acquired pneumonia is positively correlated with high survivalrates (Ahkee et. al, 1997) and maintaining a normal core bodytemperature (36.6±0.5° C.) after anesthesia decreases the incidence ofinfectious complications in patients undergoing colorectal resection(Kurz et al., 1996). Amongst the mechanisms suggested to play a role inthe antibacterial effect of heat, augmentation of the innate immunefunction is probably the most intensively studied one (Hanson, 1993;Hasday et al., 2001; Mackowiak et al., 1983; Lederman et al., 1987;Rosenpire et al., 2002). Though our results demonstrate that electricfields and sham heat application did not cause an increase in the lungtemperatures, they did raise the temperature of the peripheral organs ofthe treated mice, by at least 2 degrees (the normal skin temperature ofmice is 34-35° C. while in the present study the electrodes temperaturewas set to 37° C.). Hanson (1993) demonstrated that mature primaryT-cells response is strongly regulated by temperature changes in therange of 29° to 37° C., which corresponds to the peripheral tissuetemperature elevation during febrile episodes. This finding provides apossible explanation for the sham heat inhibitory effect reported in thepresent study. Since application of electric fields is always associatedwith heat production it is reasonable to assume that the enhancedefficacy of the fields reported in the present study compared to ourprevious report (Giladi et al., 2008) is in part due to elevatedtemperatures of the peripheral tissue.

Thus, electric fields can be an effective and safe antibacterialtreatment modality which could be applied in cases of both superficialand deep infections, either as a sole treatment or in combination withantibiotics, for both P. aeruginosa pathogens and other types ofbacteria. Such treatments will involve placement of insulated electrodeson the skin surrounding the infected organs, and generating electricfields by applying the appropriate AC voltages to those electrodes.

While the invention has been particularly shown and described withreference to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details can bemade without departing from the spirit and scope of the invention.

What is claimed is:
 1. A method of selectively destroying or inhibitingthe growth of bacteria located within a target region of a patient,comprising the steps of: administering, to the patient, an antibioticagainst the bacteria, so that a therapeutically effective dose arrivesin the target region; capacitively coupling an AC electric field intothe target region of the patient while the therapeutically effectivedose is present at the target region, wherein the electric field hasfrequency characteristics that correspond to a vulnerability of thebacteria, wherein the electric field is strong enough to damage, duringcell division, a significant portion of the bacteria whose long axis isgenerally aligned with the lines of force of the electric field, andwherein the electric field leaves non-dividing cells located within thetarget region substantially unharmed; and repeating the coupling stepuntil a therapeutically significant portion of the bacteria die, whereinthe strength of the electric field in at least a portion of the targetregion is between 2 V/cm and 100 V/cm.
 2. The method of claim 1, whereinthe strength of the electric field in at least a portion of the targetregion is between 5 V/cm and 20 V/cm.
 3. The method of claim 1, whereinthe strength of the electric field in at least a portion of the targetregion is between 10 V/cm and 17 V/cm.
 4. The method of claim 3, whereinthe frequency of the electric field is between 5 MHz and 20 MHz.
 5. Themethod of claim 3, wherein the frequency of the electric field is about10 MHz.
 6. The method of claim 1, wherein the electric field has a firstorientation during a first interval of time and a second orientationduring a second interval of time, wherein at least a portion of thefirst interval of time and the second interval of time are mutuallyexclusive.
 7. The method of claim 6, wherein the first orientation isgenerally perpendicular to the second orientation.